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Leeches are amazing animals that can be classified as conditionally poisonous animals since the salivary cocktail they produce is injected directly into the victim, and its components have strictly defined biological purposes, such as preventing blood clot formation. Thrombolytic drugs are mainly aimed at treating newly formed blood clots. Aged clots are stabilized by a large number of isopeptide bonds that prevent the action of thrombolytics. These bonds are destroyed by destabilase, an enzyme of the leech’s salivary glands. Here, we conducted a pilot study to evaluate the feasibility and effectiveness of the use of destabilase in relation to blood clots formed during real pathological processes. We evaluated the isopeptidase activity of destabilase during the formation of a stabilized fibrin clot. We showed that destabilase does not affect the internal and external coagulation cascades. We calculated the dose–response curve and tested the ability of destabilase to destroy isopeptide bonds in natural blood clots. The effect of aged and fresh clots dissolving ability after treatment with destabilase coincided with the morphological characteristics of clots during surgery. Thus, recombinant destabilase can be considered as a potential drug for the treatment of aged clots, which are difficult to treat with known thrombolytics.

The global problem of emerging resistance of microorganisms to antibiotics makes the search for new natural substances with antibacterial properties relevant. Such substances include peptidoglycan recognition proteins (PGLYRP), which are the components of the innate immunity of many organisms, including humans. These proteins have a unique mechanism of action that allows them to evade the resistance of bacteria to them, as well as to be active against both Gram-positive and Gram-negative bacteria. However, the use of antimicrobial recombinant proteins is not always advisable due to the complexity of local delivery of the proteins and their stability; in this regard it seems appropriate to activate the components of the innate immunity. The aim of this study was to increase the expression level of native peptidoglycan recognition protein genes in HeLa cells using genome-editing technology with synergistic activation mediators (CRISPR/Cas9-SAM) and evaluate antichlamydial effect of PGLYRP. We demonstrated activation of the chlamydial two-component gene system (ctcB-ctcC), which played a key role in the mechanism of action of the peptidoglycan recognition proteins. We generated the HeLa cell line transduced with lentiviruses encoding CRISPR/Cas9-SAM activation system with increased PGLYRP gene expression. It was shown that activation of the own peptidoglycan recognition proteins gene expression in the cell line caused inhibition of the chlamydial infection development. The proposed approach makes it possible to use the capabilities of innate immunity to combat infectious diseases caused by Gram-positive and Gram-negative bacteria.

Infectious bursal disease virus (IBDV), which infects young chickens, is one of the most important pathogens that harm the poultry industry. Evaluation of the immune status of birds before and after vaccination is of great importance for controlling the disease caused by this virus. Therefore, the development of low-cost and easy-to-manufacture test systems for IBDV antibody detection remains an urgent issue. In this study, three expression systems (bacteria, yeast, and human cells) were used to produce recombinant VP3 protein of IBDV. VP3 is a group-specific antigen and hence may be a good candidate for use in diagnostic tests. Comparison of the antigenic properties of the obtained polypeptides showed that the titres of antibodies raised in chickens against bacteria- or human-cell-derived recombinant VP3 were high, whereas the antibody level against yeast-derived recombinant VP3 was low. The results of an enzyme-linked immunosorbent assay (ELISA) of sera from IBDV-infected chickens demonstrated that the recombinant VP3 produced in E. coli would be the best choice for use in test systems.

Recent evidence shows that small open reading frame (smORF

The microbiota of the respiratory tract remains a relatively poorly studied subject. At the same time, like the intestinal microbiota, it is involved in modulating the immune response to infectious agents in the host organism. A causal relationship between the composition of the respiratory microbiota and the likelihood of development and the severity of COVID-19 may be hypothesized. We analyze biomaterial from nasopharyngeal smears from 336 patients with a confirmed diagnosis of COVID-19, selected during the first and second waves of the epidemic in Russia. Sequences from a similar study conducted in Spain were also included in the analysis. We investigated associations between disease severity and microbiota at the level of microbial community (community types) and individual microbes (differentially represented species). To search for associations, we performed multivariate analysis, taking into account comorbidities, type of community and lineage of the virus. We found that two out of six community types are associated with a more severe course of the disease, and one of the community types is characterized by high stability (very similar microbiota profiles in different patients) and low level of lung damage. Differential abundance analysis with respect to comorbidities and community type suggested association of Rothia and Streptococcus genera representatives with more severe lung damage, and Leptotrichia, unclassified Lachnospiraceae and Prevotella with milder forms of the disease.

In the search for anti-SARS-CoV-2 drugs, much attention is given to safe and widely available native compounds. The green tea component epigallocatechin 3 gallate (EGCG) is particularly promising because it reportedly inhibits viral replication and viral entry in vitro. However, conclusive evidence for its predominant activity is needed. We tested EGCG effects on the native virus isolated from COVID-19 patients in two independent series of experiments using VERO cells and two different treatment schemes in each series. The results confirmed modest cytotoxicity of EGCG and its substantial antiviral activity. The preincubation scheme aimed at infection prevention has proven particularly beneficial. We complemented that finding with a detailed investigation of EGCG interactions with viral S-protein subunits, including S2, RBD, and the RBD mutant harboring the N501Y mutation. Molecular modeling experiments revealed N501Y-specific stacking interactions in the RBD-ACE2 complex and provided insight into EGCG interference with the complex formation. Together, these findings provide a molecular basis for the observed EGCG effects and reinforce its prospects in COVID-19 prevention therapy.

Under severe or prolonged stress, bacteria produce a nonspecific DNA-binding protein (Dps), which effectively protects DNA against damaging agents both in vitro and in vivo by forming intracellular biocrystals. The phenomenon of protective crystallization of DNA in living cells has been intensively investigated during the last two decades; however, the results of studies are somewhat contradictory, and up to now, there has been no direct determination of a Dps-DNA crystal structure. Here, we report the in vitro analysis of the vital process of Dps-DNA co-crystallization using two complementary structural methods: synchrotron small-angle X-ray scattering in solution and cryo-electron tomography. Importantly, for the first time, the DNA in the co-crystals was visualized, and the lattice parameters of the crystalline Dps-DNA complex were determined.

Background Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. Results We annotated the Hirudo medicinalis genome and performed RNA-seq on salivary cells isolated from three closely related leech species, H. medicinalis, Hirudo orientalis, and Hirudo verbana. Differential expression analysis verified by proteomics identified salivary cell-specific gene expression, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants have been found to be expressed not only in salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis. Conclusions Here we report a genome draft of Hirudo medicinalis and describe identification of novel salivary proteins and new homologs of genes encoding known anticoagulants in transcriptomes of three medicinal leech species. Our data provide new insights in genetics of blood-feeding lifestyle in leeches.

Series of mutant genes of prokaryotic uridine phosphorylases (Shewanella oneidensis MR-1, Escherichia coli) were constructed, and the resulting strains-producers of the corresponding proteins were obtained. Proteins were purified, and their physicochemical and fermentative properties were studied. On the basis of the obtained data, the role of individual amino acid residues of the polypeptide chain of uridine phosphorylases in the formation and functioning of the phosphate-binding site in these proteins was shown. The assumption of independent binding of two substrates, ion of inorganic phosphate (Pi) and uridine (Urd), by nucleoside phosphorylases, was made.

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The rise of antibiotic resistance has necessitated the development of alternative strategies for the treatment of infectious diseases. Antimicrobial peptides (AMPs), components of the innate immune response in various organisms, are promising next-generation drugs against bacterial infections. The ability of the medicinal leech Hirudo medicinalis to store blood for months with little change has attracted interest regarding the identification of novel AMPs in this organism. In this study, we employed computational algorithms to the medicinal leech genome assembly to identify amino acid sequences encoding potential AMPs. Then, we synthesized twelve candidate AMPs identified by the algorithms, determined their secondary structures, measured minimal inhibitory concentrations against three bacterial species ( Escherichia coli , Bacillus subtilis , and Chlamydia thrachomatis ), and assayed cytotoxic and haemolytic activities. Eight of twelve candidate AMPs possessed antimicrobial activity, and only two of them, 3967 (FRIMRILRVLKL) and 536–1 (RWRLVCFLCRRKKV), exhibited inhibition of growth of all tested bacterial species at a minimal inhibitory concentration of 10 μmol. Thus, we evidence the utility of the developed computational algorithms for the identification of AMPs with low toxicity and haemolytic activity in the medicinal leech genome assembly.

Peptidoglycan recognition proteins (PGLYRPs) are innate immune components that recognize the peptidoglycan and lipopolysaccharides of bacteria and exhibit antibacterial activity. Recently, the obligate intracellular parasite Chlamydia trachomatis was shown to have peptidoglycan. However, the antichlamydial activity of PGLYRPs has not yet been demonstrated. The aim of our study was to test whether PGLYRPs exhibit antibacterial activity against C. trachomatis . Thus, we cloned the regions containing the human Pglyrp1 , Pglyrp2 , Pglyrp3 , and Pglyrp4 genes for subsequent expression in human cell lines. We obtained stable HeLa cell lines that secrete recombinant human PGLYRPs into culture medium. We also generated purified recombinant PGLYRP1, -2, and -4 and confirmed their activities against Gram-positive ( Bacillus subtilis ) and Gram-negative ( Escherichia coli ) bacteria. Furthermore, we examined the activities of recombinant PGLYRPs against C. trachomatis and determined their MICs. We also observed a decrease in the infectious ability of chlamydial elementary bodies in the next generation after a single exposure to PGLYRPs. Finally, we demonstrated that PGLYRPs attach to C. trachomatis elementary bodies and activate the expression of the chlamydial two-component stress response system. Thus, PGLYRPs inhibit the development of chlamydial infection.

Mollicutes (mycoplasmas) feature a significant loss of known regulators of gene expression. Here, we identified the recognition site of the MraZ-family regulator of Mycoplasma gallisepticum, which is conserved in many species of different clades within class Mollicutes. The MraZ binding site is AAAGTG[T/G], in the promoter of mraZ gene it forms a series of direct repeats with a structure (AAAGTG[T/G]N3)k, where k = 3 most frequently. MraZ binds to a single repeat as an octamer complex. MraZ can also bind a single binding site or a series of repeats with different spacer lengths (2–4 nt); thus, it may play a role in the regulation of multiple operons in Mollicutes. In M. gallisepticum, MraZ acts as a transcriptional activator. The overexpression of MraZ leads to moderate filamentation of cells and the formation of aggregates, likely as a result of incomplete cytokinesis.

The procedure for obtaining an active recombinant destabilase from the medicinal leech in Escherichia coli cells was developed. The plasmids encoding an analogue of native destabilase, as well as the protein forms carrying polyhistidine sequence at the Cand/or N-terminus of the polypeptide were obtained during the work. The producing strains of different forms of the protein were constructed, the cultivation process was optimized. The conditions of renaturation of destabilase recombinant forms by dialysis and using chromatographic absorbent were selected. The muramidase activity towards cell walls of Micrococcus lysodeikticus bacferia and lytic activity towards E. coli were investigated. The dependence of pH and ionic strength of the solution on the activities was determined. The total antibacterial activity of destabilase towards E. coli was shown.

Destabilase-lysozyme (mlDL) is an enzyme secreted by the salivary gland cells of medicinal leeches. Destabilase-lysozyme possesses lysozyme and isopeptidase activities. We generated recombinant destabilase-lysozyme isoform 2 in three expression systems, i.e., in the bacteria Escherichia coli, in the yeast Pichia pastoris, and in the human cell line Expi293F. In E. coli, we generated both polypeptide in inclusion bodies that was later undergone to the refolding and soluble protein that had been fused with the chaperone SlyD. The chaperone was later cleaved by a specific TEV-protease. In cultures of the yeast P. pastoris and the human cell line Expi293F, the soluble form of destabilase-lysozyme was accumulated in the culture media. For the generated enzymes, we determined the lysozyme, isopeptidase and fibrinolytic activities and tested their general antimicrobial effects. The comparisons of the enzymes generated in the different expression systems revealed that all of the destabilase-lysozymes obtained in the soluble forms possessed equal levels of lysozyme, isopeptidase and fibrinolytic activities that exceeded several to ten times the levels of the same activities of the destabilase-lysozyme renaturated from the inclusion bodies. A similar pattern of the differences in the levels of the general antimicrobial effects was observed for the destabilase-lysozymes generated in the soluble form and as inclusion bodies.

A novel strain of infectious bursal disease virus, named DD1, was isolated from broiler chickens in Russia in 2016. Here, we present its complete genome sequence., A novel strain of infectious bursal disease virus, named DD1, was isolated from broiler chickens in Russia in 2016. Here, we present its complete genome sequence. Nucleotide sequence analysis of both segments of the virus suggests that it belongs to a group of very virulent strains.

Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. We annotated theHirudo medicinalisgenome and performed RNA-seq on salivary cells isolated from three closely related leech species,H. medicinalis, Hirudo orientalis,andHirudo verbana.Differential expression analysis verified by proteomics identified salivary cell-specific genes, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants were not differentially expressed in the salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis. Thus, our study provides one of the most comprehensive knowledge of the genetic fundamentals of the blood-sucking lifestyle in leeches.

Introduction Known thrombolytic agents either break peptide bonds in the fibrin molecule or act as plasminogen activators, which also results in peptide bond cleavage. In thrombi, fibrin molecules are known to be cross-linked by isopeptide bonds, the formation of which is mediated by factor XIIIa. In this work, we studied the dissolution of thrombi via isopeptide bond cleavage using a recombinant destabilase. Destabilase is an enzyme secreted from the medicinal leech salivary gland. This enzyme exhibits muramidase (lysozyme) activity, in addition to endo-e-(γ-Glu)–Lys-isopeptidase activity, which is responsible for isopeptide bond cleavage. Methods Venous (jugular vein) and arterial (carotid artery) thrombosis was induced in rats. Rats were intravenously injected with both recombinant destabilase produced in Escherichia coli and a commercial streptokinase preparation. After 24 h, the weight and degree of cross-linking in the thrombi were analysed. Amidolytic activity in rat blood serum was measured in order to evaluate destabilase levels in the blood. Results Destabilase was definitively shown to cause a 47.6% and 74.6% decrease in the weight of venous and arterial thrombi, respectively. The enzyme proved to be more efficient at dissolving thrombi compared to streptokinase. The combined administration of destabilase and streptokinase has a greater effect than the injection of individual enzymes. Destabilase reduces fibrin stabilization in thrombi. Conclusion Cumulatively, we find that the medicinal leech destabilase is a more efficient thrombolytic agent for dissolving thrombi, which could help increase the overall effectiveness of conventional thrombolytic drugs.

Sea anemones (Actiniaria) are intensely popular objects of study in venomics. Order Actiniaria includes more than 1,000 species, thus presenting almost unlimited opportunities for the discovery of novel biologically active molecules. The venoms of cold-water sea anemones are studied far less than the venoms of tropical sea anemones. In this work, we analysed the molecular venom composition of the cold-water sea anemone Cnidopus japonicus. Two sets of NGS data from two species revealed molecules belonging to a variety of structural classes, including neurotoxins, toxin-like molecules, linear polypeptides (Cys-free), enzymes, and cytolytics. High-throughput proteomic analyses identified 27 compounds that were present in the venoms. Some of the toxin-like polypeptides exhibited novel Cys frameworks. To characterise their function in the venom, we heterologously expressed 3 polypeptides with unusual Cys frameworks (designated CjTL7, CjTL8, and AnmTx Cj 1c-1) in E. coli. Toxicity tests revealed that the CjTL8 polypeptide displays strong crustacean-specific toxicity, while AnmTx Cj 1c-1 is toxic to both crustaceans and insects. Thus, an improved NGS data analysis algorithm assisted in the identification of toxins with unusual Cys frameworks showing no homology according to BLAST. Our study shows the advantage of combining omics analysis with functional tests for active polypeptide discovery.

Here we present two incomplete mitochondrial genome sequences of Hirudo medicinalis and Hirudo verbana (Annelida, Hirudinea). The corresponding sequences are 14,729 and 14,604 base pairs in length. They contain all mitochondrial genes (13 protein-coding genes, 22 tRNAs and two rRNAs) but lack the non-coding region. Nevertheless, the robust reconstruction of their phylogenetic relationships presented here reveals distinct separation of both leeches from other annelids and at the same time relatively high dissimilarity between each other.

Fragilysin (BFT) is metalloprotease that is secreted by enterotoxigenic Bacteroides fragilis. Studying the mechanism of BFT interaction with intestinal epithelial cells requires a pure protein sample. In this study, we cloned DNA-fragments coding for the catalytic domain of fragilysin-2 and profragilysin-2 into an E. coli expression vector. Purification methods for the recombinant fragilysin-2 catalytic domain and profragilysin-2 were developed. In addition, we obtained mature active fragilysin-2 from recombinant proprotein by limited tryptic digestion. We tested the biological activity of the recombinant protein samples and revealed that E-cadherin was cleaved when HT-29 cells were treated with mature fragilysin-2 but not with profragilysin-2. Azocoll, azocasein and gelatin were not proteolytically cleaved by mature fragilysin-2. Proteins released in culture medium after HT-29 cells treatment with mature active BFT-2 were identified.

A DNA fragment containing a promoter-operator and structural parts of the uridine phosphorylase gene from Shewanella oneidensis MR-1 was cloned. Cross-heterological expression of the udp genes from Sh. oneidensis MR-1 and Escherichia coli under the control of authentic regulatory regions is shown. The UDP protein accumulates in an active form in the cytoplasmic fraction of cells. The recombinant UDP protein from Sh. oneidensis MR-1 obtained by heterological expression was isolated and characterized. E. coli udp gene promoter activity was observed during heterological expression in Sh. oneidensis MR-1 cells under both aerobic and anaerobic conditions.

Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The structure of the C212S mutant of uridine phosphorylase from the facultatively aerobic Gram-negative γ-proteobacteriumShewanella oneidensisMR-1 (SoUP) was determined at 1.68 Å resolution. A comparison of the structures of the mutant and the wild-type enzyme showed that one dimer in the mutant hexamer differs from all other dimers in the mutant and wild-type SoUP (both in the free form and in complex with uridine). The key difference is the `maximum open' state of one of the subunits comprising this dimer, which has not been observed previously for uridine phosphorylases. Some conformational features of the SoUP dimer that provide access of the substrate into the active site are revealed. The binding of the substrate was shown to require the concerted action of two subunits of the dimer. The changes in the three-dimensional structure induced by the C212S mutation account for the lower affinity of the mutant for inorganic phosphate, while the affinity for uridine remains unchanged.

α-Crystallin-type small heat shock proteins (sHsps) are expressed in many bacteria, animals, plants, and archaea. Among mycoplasmas (Mollicutes), predicted sHsp homologues so far were found only in the Acholeplasmataceae family. In this report, we describe the cloning and functional characterization of a novel sHsp orthologue, IbpA protein, present in Acholeplasma laidlawii. Importantly, similar to the endogenously expressed sHsp proteins, the recombinant IbpA protein was able to spontaneously generate oligomers in vitro and to rescue chemically denatured bovine insulin from irreversible denaturation and aggregation. Collectively, these data suggest that IbpA is a bona fide member of the sHsps family. The immune-electron microscopy data using specific antibodies against IbpA have revealed different intracellular localization of this protein in A. laidlawii cells upon heat shock, which suggests that IbpA not only may participate in the stabilization of individual polypeptides, but may also play a protective role in the maintenance of various cellular structures upon temperature stress.

Enterotoxin A containing various leader sequences have been obtained by site-driven mutagenesis. Some of them were capable of providing the translocation of recombinant SEA to E. coli periplasmic space. Structure of C-region of the signal peptide is essential for intracellular protein location. Substitution of a more common to E. coli proproteins Ala-Ser-Ala for the authentic sequence of Val-Asn-Gly is the most important in recognition by signal peptidase type I.

Background Destabilase-Lysozyme (mlDL) is a multifunctional i-type enzyme that has been found in the secretions from the salivary glands of medicinal leeches. mlDL has been shown to exhibit isopeptidase, muramidase and antibacterial activity. This enzyme attracts interest because it expresses thrombolytic activity through isopeptidolysis of the ε-(γ-Glu)-Lys bonds that cross-link polypeptide chains in stabilised fibrin. To date, three isoforms of mlDL have been identified. The enzymatic properties of pure mlDL isoforms have not yet been described because only destabilase complexes containing other proteins could be isolated from the salivary gland secretion and because low product yield from the generation of recombinant proteins has made comprehensive testing difficult. Results In the present study, we optimised the procedures related to the expression, isolation and purification of active mlDL isoforms (mlDL-Ds1, mlDL-Ds2, mlDL-Ds3) using an Escherichia coli expression system, and we detected and compared their muramidase, lytic, isopeptidase and antimicrobial activities. After optimisation, the product yield was 30 mg per litre of culture. The data obtained in our study led to the suggestion that the recombinant mlDL isoforms isolated from inclusion bodies form stable oligomeric complexes. Analyses of the tested activities revealed that all isoforms exhibited almost identical patterns of pH and ionic strength effects on the activities. We determined that mlDL-Ds1, 2, 3 possessed non-enzymatic antibacterial activity independent of their muramidase activity. For the first time, we demonstrated the fibrinolytic activity of the recombinant mlDL and showed that only intact proteins possessed this activity, suggesting their enzymatic nature. Conclusions The recombinant Destabilase-Lysozyme isoforms obtained in our study may be considered potential thrombolytic agents that act through a mechanism different from that of common thrombolytics. Electronic supplementary material The online version of this article (doi:10.1186/s12858-015-0056-3) contains supplementary material, which is available to authorized users.

Here we present the complete genome sequence of Bacteroides fragilis isolate BOB25. It is an enterotoxigenic isolate that was obtained from a stool sample of a patient with dysbiosis.

The fragilysin (BFT) is a protein secreted by enterotoxigenic Bacteroides fragilis strains. BFT contains zinc-binding motif which was found in the metzincins family of metalloproteinases. In this study, we generated three known recombinant isoforms of BFT using Escherichia coli, tested their activity and examined whether E-cadherin is a substrate for BFTs. BFT treatment of HT-29 cells induced endogenous E-cadherin cleavage, and this BFT activity requires the native structure of zinc-binding motif. At the same time recombinant BFTs did not cleave recombinant E-cadherin or E-cadherin in isolated cell fractions. It indicates that E-cadherin may be not direct substrate for BFT. We also detected and identified proteins released into the cultural medium after HT-29 cells treatment with BFT. The role of these proteins in pathogenesis and cell response to BFT remains to be determined.

Uridine phosphorylase (UDP, EC 2.4.2.3), a key enzyme in the pyrimidine salvage pathway, catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The gene expression of UDP fromShewanella oneidensisMR-1 was performed in the recipient strainEscherichia coli. The UDP protein was crystallized on earth (in the free form and in complex with uridine as the substrate) by the hanging-drop vapour-diffusion method at 296 K and under microgravity conditions (in the free form) aboard the Russian Segment of the International Space Station by the capillary counter-diffusion method. The data sets were collected to a resolution of 1.9 Å from crystals of the free form grown on earth, 1.6 Å from crystals of the complex with uridine and 0.95 Å from crystals of the free form grown under microgravity. All crystals belong to the space groupP21and have similar unit-cell parameters. The crystal of uridine phosphorylase grown under microgravity diffracted to ultra-high resolution and gave high-quality X-ray diffraction data.

Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Expression of UP fromShewanella oneidensisMR-1 (SoUP) was performed inEscherichia coli. The high-resolution X-ray structure of SoUP was solved in the free form and in complex with uridine. A crystal of SoUP in the free form was grown under microgravity and diffracted to ultrahigh resolution. Both forms of SoUP contained sulfate instead of phosphate in the active site owing to the presence of ammonium sulfate in the crystallization solution. The latter can be considered as a good mimic of phosphate. In the complex, uridine adopts a high-synconformation with a nearly planar ribose ring and is present only in one subunit of the hexamer. A comparison of the structures of SoUP in the free form and in complex with the natural substrate uridine showed that the subunits of the hexamer are not identical, with the active sites having either an open or a closed conformation. In the monomers with the closed conformation, the active sites in which uridine is absent contain a glycerol molecule mimicking the ribose moiety of uridine.

The vertical growth stage is the most dangerous stage of melanoma and is often associated with a poor prognosis. The increased invasiveness and metastasis that is typical for vertically growing melanoma are mediated by the molecules of cell adhesion (particularly, integrins). Integrin αvβ3, which is abundantly expressed on melanoma cells with high metastatic potentials and is characterized by low expression levels in normal melanocytes, is potentially an attractive target for melanoma diagnostics and therapy. Integrin αvβ3 is known to recognize the arginine-glycine-aspartic (RGD) sequence, which has been found in a wide variety of its natural ligands. Here expression vectors bearing the genes of fusion proteins have been constructed for producing these proteins in Escherichia coli. Such fusion proteins consist of a peptidic 'address,' targeting the integrins on melanoma cells, linked to an 'adaptor' for the attachment of a diagnostic or toxic agent. The peptidic 'address' contains the RGD motif, which is stabilized by a disulfide bond to achieve the optimal receptor binding conformation. The 'adaptor' is a tetrameric protein, namely, streptavidin, that is able to achieve high-affinity binding of d-biotin (K(d) = 10(-15) M) and confer avidity to the address peptide. This binding ability facilitates the generation of anti-melanoma diagnostic and therapeutic agents using the appropriate biotin derivatives. These recombinant proteins were purified from the periplasm of E.coli using columns with 2-iminobiotin agarose and demonstrated an ability to adhere to the surface of murine and human melanoma cells.

Background Bacteria of the class Mollicutes underwent extreme reduction of genomes and gene expression control systems. Only a few regulators are known to date. In this work, we describe a novel group of transcriptional regulators that are distributed within different Mollicutes and control the expression of restriction-modification systems (RM-systems). Results We performed cross-species search of putative regulators of RM-systems (C-proteins) and respective binding sites in Mollicutes. We identified a set of novel putative C-protein binding motifs distributed within Mollicutes. We studied the most frequent motif and respective C-protein on the model of Mycoplasma gallisepticum S6. We confirmed our prediction and identified key nucleotides important for C-protein binding. Further we identified novel target promoters of C-protein in M. gallisepticum. Conclusions We found that C-protein of M. gallisepticum binds predicted conserved direct repeats of the (GTGTTAN5)2 motif. Apart from its own operon promoter, HsdC can bind to the promoters of the clpB chaperone gene and a tRNA cluster. Electronic supplementary material The online version of this article (doi:10.1186/s12866-017-0935-4) contains supplementary material, which is available to authorized users.