Effect of Heterologous Expression of Chemotaxis Proteins from Genus Thermotoga on the Growth Kinetics of Escherichia coli Cells.

In the process of optimization of heterologous expression of thermostable chemotaxis proteins CheW and CheY as industrially useful polypeptides, their direct influence on the cell growth kinetics and morphology of Escherichia coli was observed. CheW and CheY of bacteria of the genus Thermotoga, being expressed in recombinant form in E. coli cells, are involved in the corresponding signal pathways of the mesophilic microorganisms. The effects of such involvement in the metabolism of "host" cells are extremely diverse: from rapid aging of the culture to elongation of the stationary growth phase. We also discuss the mechanisms of the influence of the heterologous chemotaxis proteins on cells, their positive and negative effects, as well as potential applications in industry and biomedicine.

Thermostable Recombinant Polypeptides as the Source of L-Amino Acids for Culture Media.

Mutant homologues of small chemotactic and DNA-binding proteins from thermophilic bacteria Thermotoga petrophila RKU-1 and Thermotoga naphthophila were obtained. These proteins can be expressed in the recombinant form in E. coli cells. A wide range of properties and parameters that are important for isolation of these proteins were revealed: stability in a wide temperature and pH range, high level of expression, solubility, and the possibility of using simple purification schemes with low number of successive steps. The positive effect of proteins on in vitro fibroblasts growth was demonstrated. The described properties of the target proteins indicate the possibility of their use in different biotechnology industries as an inexpensive source of L-amino acids.

Induction of Alternative Splicing and Inhibition of Activity of Telomerase Catalytic Subunit by Apoptotic Endonuclease EndoG in Human T, B, and NK Cells.

The effect of apoptotic endonuclease EndoG on alternative splicing of mRNA of human telomerase catalytic subunit hTERT (human telomerase reverse transcriptase) and telomerase activity in normal human lymphocytes were studied. Human CD4+, CD8+, B, and NK cells were transfected with a plasmid pEndoG-GFP containing EndoG gene or control plasmid pGFP. The levels of mRNA of EndoG or hTERT splicing variants were analyzed by real-time PCR. Protein content was assessed by Western blotting. Telomerase activity was measured by the telomere repeats amplification protocol. EndoG overexpression reduced the expression of active full-length hTERT and increased the expression of inactive splice variant. Shifted balance of hTERT splice variants in cells led to a significant decrease in telomerase activity within 72 h after transfection.

Apoptotic Endonuclease EndoG Inhibits Telomerase Activity and Induces Malignant Transformation of Human CD4+ T Cells.

Telomerase activity is regulated by an alternative splicing of mRNA of the telomerase catalytic subunit hTERT (human telomerase reverse transcriptase). Increased expression of the inactive spliced hTERT results in inhibition of telomerase activity. Little is known about the mechanism of hTERT mRNA alternative splicing. This study was aimed at determining the effect of an apoptotic endonuclease G (EndoG) on alternative splicing of hTERT and telomerase activity in CD4+ human T lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of the active full-length hTERT variant and upregulated the inactive alternatively spliced variant. Reduction of full-length hTERT levels caused downregulation of the telomerase activity, critical telomere shortening during cell division that converted cells into the replicative senescence state, activation of apoptosis, and finally cell death. Some cells survive and undergo a malignant transformation. Transformed cells feature increased telomerase activity and proliferative potential compared to the original CD4+ T cells. These cells have phenotype of T lymphoblastic leukemia cells and can form tumors and cause death in experimental mice.

Intracellular Localization of Apoptotic Endonuclease EndoG and Splice-Variants of Telomerase Catalytic Subunit hTERT.

The activity of telomerase catalytic subunit hTERT (human telomerase reverse transcriptase) can be regulated by alternative splicing of its mRNA. The mechanism of hTERT splicing is not understood in detail. Apoptotic endonuclease EndoG is known to participate in this process. In the present work, the intracellular colocalization and mRNA levels of EndoG and hTERT splice-variants in normal and apoptotic cancer cells were studied. We found that the development of apoptosis increased the expression of EndoG and changed the ratio of hTERT splice-variants, which decreased the telomerase activity in the cells. The development of apoptosis was accompanied by changes in the amount of mRNA and in the localization of EndoG and hTERT splice-variants in the cytoplasm, nuclei, and mitochondria of the cells. The suppression of EndoG expression using RNA interference prevented induction of the α+ß- splice-variant of hTERT and inhibition of the telomerase activity. A high degree of the intracellular colocalization of EndoG and hTERT was shown. The changes in the expression and localization of EndoG corresponded with changes in the amount and localization of hTERT splice-variants. These data confirm the participation of EndoG in the alternative splicing of mRNA of the telomerase catalytic subunit and in regulation of the telomerase activity.

[PEGylated recombinant L-asparaginase from Erwinia carotovora: Production, properties, and potential applications].

N-hydroxysuccinimide ester of monomethoxy polyethylene glycol hemisuccinate was synthesized. It acylated amino groups in a molecule of recombinant L-asparaginase from Erwinia carotovora. A method of L-asparaginase modification by the obtained activated polyethylene glycol derivative was developed. The best results were produced by modification of the enzyme with a 25-fold excess of reagent relative to the enzyme tetramer. The modified L-asparaginase was isolated from the reaction mixture by gel filtration on Sepharose CL-6B. The purified bioconjugate did not contain PEG unbound to the protein, demonstrated high catalytic activity, and exhibited antiproliferative action on cell cultures.

[Bioactive proteins and peptides: current state and new trends of practical application in the food industry and feed production].

Proteins and polypeptides play a key role in the life of a human body. Scientific and practical interest to the natural proteinaceous substances could be explained by the diversity of their functions in metabolic processes. Biologically active substances of the protein origin have a rich history of applications in different sectors of the economy. In this case, the close relationship is observed between food industry, biomedicine and fodder production, because efficient conversion of feed protein in productive agricultural animals provides, as a result, the required level of metabolism in a human, as the main consumer of final products derived from these animals. Obviously, for normal growth, development and resistance to infectious agents, both people and farm animals need a constant consumption of L-amino acids in certain proportions and in available for absorption form. This review considers the bioactive polypeptides used in nutrition and food industry, main trends and practical approaches to generating protein products with the desired characteristics.

[Physicochemical properties and antiproliferative activity of recombinant Yersinia pseudotuberculosis L-asparaginase].

The physicochemical, catalytic, and antiproliferative activity of a recombinant L-asparaginase from Yersinia pseudotuberculosis (YpA) have been studied. The following results were obtained: the K(M) value for L-asparagine is 17 +/- 0.9 microM, the optimal temperature is 60 degrees C, pH is 8.0, pI is 5.4 +/- 0.3, the L-glutaminase activity is no more than 5-6% of the L-asparaginase activity, and the antiproliferative activity on the Fisher L5178y lymphadenosis cell line comprised T/C = 136% (p < 0.001) at a 15% recovery rate. The described characteristic allows one to regard YpA as an antitumor enzyme with biological features similar to the L-asparaginase of E. coli.

Cloning, expression and characterization of the recombinant Yersinia pseudotuberculosis L-asparaginase.

We have cloned ansB (YPTB1411) gene from Yersinia pseudotuberculosis Q66CJ2 and constructed stable inducible expression system that overproduce L-asparaginase from Y. pseudotuberculosis (YpA) in Escherichiacoli BL21 (DE3) cells. For purification of YpA we used Q-Sepharose and DEAE-Toyopearl column chromatography. We examined kinetics of the enzyme reaction, catalytic activity as a function of pH, temperature and ionic strength, thermostability and other enzyme properties. Biochemical properties of YpA are similar with those of E. coli type II L-asparaginase. K(m) for L-asparagine is 17 ± 0.9 µM and pI 5.4 ± 0.3. Enzyme demonstrates maximum activity at pH 8.0 and 60 °C. YpA L-glutaminase activity is relatively low and more than 15 times less than specific activity towards L-asn. We evaluated also the antiproliferative effect of YpA in vitro and in vivo with E. colil-asparaginase (EcA) as the reference substance at similar conditions.

[Mechanisms of development of side effects and drug resistance to asparaginase and ways to overcome them]. / Mekhanizmy razvitiia pobochnykh éffektov i lekarstvennoi ustoichivosti k asparaginazam i puti ikh preodoleniia.

Asparaginase is one of the most important chemotherapeutic agents against acute lymphoblastic leukemia, the most common form of blood cancer. To date, both asparaginases from E. coli and Dickeya dadantii (formerly known as Erwinia chrysanthemi), used in hematology, induce chemoresistance in cancer cells and side effects in the form of hypersensitivity of immune reactions. Leukemic cells may be resistant to asparaginase due to the increased activity of asparagine synthetase and other mechanisms associated with resistance to asparaginase. Therefore, the search for new sources of L-asparaginases with improved pharmacological properties remains a promising and prospective study. This article discusses the mechanisms of development of resistance and drug resistance to L-asparaginase, as well as possible ways to overcome them.

Creation of a producent, optimization of expression, and purification of recombinant Yersinia pseudotuberculosis L-asparaginase.

Recombinant E. coli strain producing Y. pseudotuberculosis Q66CJ2 (YpA) L-asparaginase II was created. Gene ansB homologue encoding Y. pseudotuberculosis IP 32953 L-asparaginase precursor was synthesized. The gene was cloned in pBad24 expression vector and expressed in E. coli BL21 (DE3) strain. Optimal conditions for the producer strain culturing were selected. An effective method for isolation and purification of the enzyme by two-staged column chromatography was developed.

[Differential medium for selection of bacterial L-asparaginase producer strains].

A specific, fast, and easy method for revelation of active plate producers of L-asparaginase using differential medium on the basis of LB or M9 with 1.5% agar was developed. Each 100 ml of LB or M9 medium additionally contained 6-7 ml ofglycerol, 4 g of L-asparagine, 0.2 g of CaCO3, and diagnostic components: 3 ml of 0.2 M CuSO4 x 5H2O and 2.5 ml of 0.1 M K3Fe(CN)6, pH 7.6-7.8. The results were counted 12-20 or 24-48 h after strain growth at 37 degrees C in corresponding mediums. Red color of colonies and colored zone around them showed the ability of the strain under study to destroy asparaginic complexes. The recommended method allows revealing bacterial strains producing L-asparaginase with specific activity of not less than 0.1-3.0 MU/mg of protein.

[L-asparaginases of extremophilic microorganisms in biomedicine]. / L-asparaginazy ékstremofil'nykh mikroorganizmov v biomeditsine.

L-asparaginase is extensively used in the treatment of acute lymphoblastic leukemia and several other lymphoproliferative diseases. In addition to its biomedical application, L-asparaginase is also of prospective use in food industry to reduce the formation of acrylamide, which is classified as probably neurotoxic and carcinogenic to human, and in biosensors for determination of L-asparagine level in medicine and food chemistry. The importance of L-asparaginases in different fields, disadvantages of commercial ferments, and the fact that they are widespread in nature stimuli the search for biobetter L-asparaginases from new producing microorganisms. In this regard, extremofile microorganisms exhibit unique physiological properties such as thermal stability, adaptability to extreme cold conditions, salt and pH tolerance and so provide one of the most valuable sources for novel L-asparaginases. The present review summarizes the recent results on studying the structural, functional, physicochemical and kinetic properties, stability of extremophilic L-asparaginases in comparison with their mesophilic homologues.

[Preparation and characterization of a new mutant homolog of chemotaxis protein CheY from anaerobic hyperthermophilic microorganism Thermotoga naphthophila]. / Poluchenie i kharakteristika novogo mutantnogo gomologa khemotaksisnogo belka CheY iz gipertermofil'nogo anaérobnogo mikroorganizma Thermotoga naphthophila.

Using genetic engineering methods the expression vectors structures have been designed to produce recombinant proteins TnaCheY and Tna CheY-mut, the homologues of the chemotaxis protein CheY from the hyperthermophilic organism Thermotoga naphthophila in Escherichia coli BL21(DE3) cells. The cultivation conditions of transformed strains were optimized. The influence of episomal expression of the heterologous chemotaxis protein CheY on growth kinetics parameters of the culture of mesophilic bacteria E. coli was studied. The optimal purification flowchart of the obtained proteins using thermolysis is proposed. Using the E. coli BL21(DE3) laboratory strain as an example, the possibility of employment the episomal expression of such proteins to control the cultivation and production time of pharmaceutically and industrially valuable metabolites due to the impact on some stages of the bacterial chemotaxis is experimentally proved.

[Construction and characterization of a recombinant mutant homolog of the CheW protein from Thermotoga petrophila RKU-1]. / Poluchenie i kharakteristika rekombinantnogo mutantnogo gomologa belka CheW iz gipertermofil'nogo mikroorganizma Thermotoga petrophila RKU-1.

In the work a recombinant chemotaxis protein CheW from Thermotoga petrophila RKU-1 (TpeCheW) and its mutant homolog (TpeCheW-mut) were created. It was shown that, despite the low homology with CheW prototypes from intestinal bacteria, these proteins didn't cause metabolic overload and were well expressed by cells of E. coli laboratory strains. We have discovered a broad spectrum of industrial valuable properties of the TpeCheW-mut protein such as stability in a wide range of temperatures and pH, high expression level, solubility and possibility of the application of a simple low-stage purification methodology with the use of preliminary heat treatment. Possible directions of the scientific and industrial application of this protein were claimed.

[Induction of apoptotic endonuclease EndoG with DNA-damaging agents initiates alternative splicing of telomerase catalytic subunit hTERT and inhibition of telomerase activity hTERT in human CD4+ and CD8+ T-lymphocytes]. / Induktsiia apoptoticheskoi éndonukleazy EndoG povrezhdaiushchimi DNK agentami vyzyvaet al'ternativnyi splaising kataliticheskoi sub"edinitsy telomerazy hTERT i ingibirovanie aktivnosti telomerazy v CD4+ I CD8+ T-limfotsitakh cheloveka.

Activity of telomerase catalytic subunit hTERT (human Telomerase Reverse Transcriptase) can be regulated by alternative splicing of its mRNA. At present time exact mechanism of hTERT splicing is not fully understood. Apoptotic endonuclease EndoG is known to participate this process. EndoG expression is induced by DNA damages. The aim of this work was to investigate the ability of DNA-damaging agents with different mechanism of action to induce EndoG expression and inhibit telomerase activity due to the activation of hTERT alternative splicing in normal activated human CD4+ and CD8+ T-lymphocytes. All investigated DNA-damaging agents were able to induce EndoG expression. Cisplatin, a therapeutic compound, producing DNA cross-links induced the highest level of DNA damages and EndoG expression. Incubation of CD4+ and CD8+ T-cells with cisplatin caused the changes in proportion of hTERT splice variants and inhibition of telomerase activity.

[Cisplatin-induced apoptotic endonuclease EndoG inhibits telomerase activity and causes malignant transformation of human CD4+ T lymphocytes]. / Indutsirovannaia tsisplatinom ékspressiia apoptoticheskoi éndonukleazy EndoG vyzyvaet ingibirovanie aktivnosti telomerazy i zlokachestvennuiu transformatsiiu CD4+ T-limfotsitov cheloveka.

Alternative splicing of telomerase catalytic subunit hTERT pre-mRNA (human Telomerase Reverse Transcriptase) regulates telomerase activity. Increased expression of non-active splice variant hTERT results in inhibition of telomerase. Apoptotic endonuclease EndoG is known to participate in hTERT alternative splicing. Expression of EndoG can be induced in response to DNA damages. The aim of this study was to determine the ability of a DNA-damaging compound, cisplatin, to induce EndoG and its influence on alternative splicing of hTERT and telomerase activity in human CD4+ Т lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of active full-length hTERT variant and upregulated its non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. Few cells survived and underwent malignant transformation. Transformed cells have increased telomerase activity and proliferative potential compare to initial CD4+ T cells. These cells have phenotype of T lymphoblastic leukemic cells and are able to form tumors and cause death in experimental mice.

[Suppression of telomerase activity leukemic cells by mutant forms of Rhodospirillum rubrum L-asparaginase]. / Podavlenie aktivnosti telomerazy leikoznykh kletok mutantymi formami L-asparaginazy Rhodospirillum rubrum.

The active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA): RrA+N17, D60K, F61L, RrA+N17, A64V, E67K, RrA+N17, E149R, V150P, RrAE149R, V150P and RrAE149R, V150P, F151T were obtained by the method of site-directed mutagenesis. It is established that variants RrA-N17, E149R, V150P, F151T and RrАE149R, V150P are capable to reduce an expression hTERT subunit of telomerase and, hence, activity of telomeres in Jurkat cells, but not in cellular lysates. During too time, L-asparaginases of Escherichia coli, Erwinia carotovora and Wolinella succinogenes, mutant forms RrА+N17, D60K, F61L and RrА+N17, A64V, E67K do not suppress of telomerase activity. The assumption of existence in structure RrA of areas (amino acids residues in the position 146-164, 1-17, 60-67) which are responsible for suppression of telomerase activity is made. The received results show that antineoplastic activity of some variants RrA is connected both with reduction of concentration of free L-asparagine, and with expression suppression of hTERT telomerase subunit, that opens new prospects for antineoplastic therapy.

[Prediction of protein thermostability from their primary structure: the current state and development factors]. / Prognozirovanie termostabil'nosti belkov po ikh pervichnoi strukture: sovremennoe sostoianie i faktory razvitiia.

The construction of proteins and peptides with desired properties, including resistance to high temperatures, as well as optimization of their amino acid composition, is an important and complex task, which attracts much attention in various branches of the basic sciences, and also in biomedicine and biotechnology. This raises the question: what method is more relevant for the at the pilot stage of research in order to estimate the influence of the planned amino acid substitutions on the thermostability of the resultant protein construct? In this brief review we have classified existing basic practical and theoretical approaches used in studies and predicting the thermal stability of native and recombinant polypeptides. Particular attention has been paid to the predictive potential of statistical methods for studying the thermodynamic parameters of the primary protein structure and prospects of their use.

[Apoptotic endonuclease EndoG induces alternative splicing of telomerase catalytic subunit hTERT and death of tumor cells]. / Éndonukleaza EndoG indutsiruet al'ternativnyi splaising kataliticheskoi sub"edinitsy telomerazy hTERT i gibel' opukholevykh kletok.

Telomerase activity is known to be regulated by alternative splicing of its catalytic subunit hTERT (human Telomerase Reverse Transcriptase) mRNA. Induction of non-active spliced hTERT leads to inhibition of telomerase activity. However, very little is known about the mechanism of hTERT mRNA alternative splicing. The aim of this study was to determine the role of apoptotic endonuclease EndoG in alternative splicing of hTERT and telomerase activity. Strong correlation was found between expression of EndoG and hTERT splice-variants in 12 colon cancer cell lines. Overexpression of EndoG in СаСо-2 cells downregulated the expression of active full-length hTERT variant and upregulated non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, dramatically shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. These data indicated the participation of EndoG in alternative splicing of mRNA of telomerase catalytic subunit, regulation of telomerase activity and cell fate.