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.

The activity of novel uncharacterized extremophilic L-asparaginases from the psychrophilic fungi Sclerotinia borealis , the thermoacidophilic crenarchea Acidilobus saccharovorans, and the thermophilic bacteria Melioribacter roseus were studied. Active enzymes were produced via the expression of the native L-asparaginase gene from M. roseus (MrA) and synthetic genes encoding fungal S. borealis (SbA) and archeal A. saccharovorans (AsA) L-asparaginases after codon optimization in Escherichia coli cells. In the study, the maximum specific activity at different temperatures and pH was observed for MrA. The activity of MrA crude extract was highest at 75°С and a pH of 9.0. Metal ions (1 mM) differed in their effects on enzyme activity. Сu 2+ and Zn 2+ ions completely abolished enzyme activity. Changes in the specific activity of MrA crude extract in the presence of Fe 3+ , Ni 2+ , Ca 2+ , and Mg 2+ varied within 5–28%. Our findings show that L-asparaginase of M. roseus may be a promising object for the further study of enzymatic properties and biotechnological applications.

The aim of this work was to identify genes whose mRNAs were subjected to alternative splicing by apoptotic endonuclease EndoG in CD4+ T lymphocytes from healthy humans, mice, and rats. In order to induce EndoG, lymphocytes were transfected with an EndoG-containing plasmid, or a control pGFP plasmid, or were incubated with cisplatin. Efficiency of transfection, number of cells with DNA damages and the level of EndoG expression have been monitored. Total cell mRNA has been sequenced and the changes in proportion of splice variants of genes were analyzed. The changes in the proportion of 28 mRNA splice variants have been identified in human and murine lymphocytes in both transfected with EndoG gene or incubated with cisplatin. Thus, EndoG can be considered as a potent modulator of alternative splicing of mRNA of identified genes.

— L-asparaginase is widely used in the treatment of acute lymphoblastic leukemia and several other lymphoproliferative diseases. In addition to its biomedical application, L-asparaginase is used in food industry to reduce the level of acrylamide, which is considered as neurotoxic and carcinogenic agent to humans, and in biosensors for determination of the L-asparagine level in biochemistry and food chemistry. In view of great significance of L-asparaginases in different fields, disadvantages of commercial enzymes, and the wide distribution of the enzyme in nature there is a need for novel L-asparaginases from new sources. In this context, extremophilic microorganisms exhibiting unique physiological properties such as thermal stability, adaptation to extreme cold conditions, salt, and pH tolerance attract much interest as one of the most valuable sources for novel L-asparaginases. The results of of structural, functional studies, physico-chemical properties, kinetic characteristics, and stability of L-asparaginases from extremophilic microorganisms suggest the prospect of using these enzymes in biology and medicine.

Information accumulated over the past decades on the physiological functions and metabolic pathways of biosynthesis and degradation of D-amino acids has led to a renewed interest in their study. These isomers are known to form both in nature and during the chemical synthesis of L-amino acids for feeding and pharmacological purposes, as well as in the industrial processing of some raw materials. This article discusses the positive and negative effects of D-amino acids on the human body, animals and the environment. In addition, the scientific data concerning the mechanisms of cytotoxic action of D-amino acids and their industrial and biomedical potential are summarized.

Caspase-2 is a key enzyme thinvolved in induction of apoptosis. The caspase-2 level is regulated by alternative splicing (AS) of its mRNA. The aim of this work was to determine the ability of an oligonucleotide complementary to Casp-2 pre-mRNA to induce AS. This oligonucleotide blocked the binding of splicing-regulating proteins to their sites at the end of exon 9 of Casp-2 pre-mRNA, leading to induction of AS of Casp-2 mRNA. The decrease in expression of full-size active splice-variant (Casp-2L) and the increase the expression of a shortened variant (Casp-2S) was demonstrated in human T-cell lymphoma Jurkat cell line. The expression level of total Casp-2 remained unchanged. Disproportion of splice variants of Casp-2 led to inhibition of enzymatic activity of caspase-2.

Normal human CD4+ T-lymphocytes can undergo malignant transformation during prolong cultivation in conditions of high endonuclease G (EndoG) expression or after DNA damage. The aim of this work was to study biochemical and cytogenetic features of transformed ex vivo human malignant CD4+ T- lymphocytes, as well as biochemical and morphological characteristics of tumors that develop in athymic mice after transplantation of these cells. The telomerase activity was higher and telomere length was shorter in tumor cells than in control cells. Transformed malignant cells exhibited a high level of chromosomal aberrations. Expression of genes regulating the cell cycle changed in transformed malignant CD4+ T-lymphocytes and tumor cells. The tumors that developed were classified as multicomponent Т-cell lymphomas and panniculitis-like T-cell lymphomas. Thus, transformed CD4+ T-lymphocytes can generate malignant tumors of various histogenesis.

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.

— Using genetic engineering methods we have developed expression vectors for synthesis of recombinant proteins TnaCheY and TnaCheY-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 cell 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 investigated. The optimal purification flowchart of the obtained proteins using thermolysis has been proposed. Based on the data obtained, we discuss potential areas of application of recombinant variants of the CheY thermostable chemotactic protein. Using the E. coli BL21(DE3) laboratory strain as an example, the possibility of employment of 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 has been experimentally proven.

Rru_A3730 protein is a bacterial Rhodospirillum rubrum L-asparaginase (RrA), which is known by its anticancer activity. RrA variants with point amino acid substitutions in the region of 150 amino acids residues: RrA17N, K149E, RrAE149R, V150P, F151T, RrА17N, E149R, V150P, RrAE149R, V150P, showed antiproliferative properties, and also by their ability to suppress telomerase activity. This work is devoted to comparison of physical-chemical and catalytic properties of these mutant forms of RrA. It is shown that pH optimum is in the alkaline zone (8.5 – 9.3); L-glutaminase and D-asparaginase activity is respectively not more than 0.1% and 1.6% of L-asparaginase for all studied variants of RrA. The presence of the N17-terminal amino acid sequence MASMTGGQMGRGSSRQ of the capsid protein of bacteriophage T7 in the RrA structure leads to an increase in the thermal stability of mutant RrA analogues (from 50°C to 56°C) and their resistance to denaturation in the presence of 3 – 4 M urea. It is of Metal ions exhibit multidirectional effects on L-asparaginase activity of RrA. K+, Ca2+, Zn2+, Cs+, Co2+ in significantly affect the activity of L-asparaginase, while Mn2+, Cu2+, Fe3+ ions inhibit it. There was no correlation between antitelomerase (antiproliferative) activity and kinetic properties of mutant forms of L-asparaginase RrA.

It is known that apoptotic endonuclease G (EndoG) induces alternative splicing (AS) of telomerase catalytic subunit TERT (telomerase reverse transcriptase) mRNA and inhibits telomerase activity in tumor cells and activated human T cells. The aim of this study was to investigate the possibility of TERT mRNA AS induction and inhibition of telomerase activity by EndoG in activated mouse and rat lymphocytes. To induce EndoG expression, mouse and rat CD4 + , CD8 + T cells, B cells, and NK cells were transfected with the pEndoG-GFP plasmid or incubated with the DNA-damaging agent cisplatin in vitro. The increase in the EndoG expression resulted in decreased expression of full-length active TERT variant, enhanced synthesis of the truncated splicing variant, and decreased telomerase activity. An increase in the EndoG expression, a change in the mRNA pool of TERT splicing variants, and inhibition of telomerase activity were observed in mouse and rat lymphocytes after cisplatin administration in vivo. Thus, EndoG is capable of inducing TERT mRNA AS and regulating telomerase activity in mouse and rat lymphocytes.

A new approach for the regulation of catalytic properties of the medically significant enzyme L-asparaginase is suggested based on the formation of conjugates with PEG-chitosan (chitoPEGylation). The efficiency of this approach is demonstrated using recombinant L-asparaginase from Rhodospirillum rubrum (RrA). This preparation is immunologically different from the one used in medical practice preparations of L-asparaginase from E. coli, which offers a promising alternative for applications in the case of hypersentsitivity development. The low level of activity of RrA towards L-glutamine, which decreases significantly the chance of side effects developing, is an advantage of RrA. The technique for the synthesis of the RrA conjugates with PEG-chitosan (chitoPEGylation) of a varying modification degree is developed. It is established that conjugation of RrA with PEG-chitosan increased the specific activity of the enzyme in comparison with the native one. The activity changes from 56 IU/mg (for the native enzyme) to 61–72 IU/mg (for the conjugates) depending on the degree of chitosan PEGylation. The secondary structure of the Rhodospirillum rubrum asparaginase conjugates with PEG-chitosan is examined using CD- and IR-spectroscopy. It is found that the enzyme structure changed only slightly as a result of conjugation with PEG-chitosan: the content of α-helices changed from 36% (for the native enzyme) to 30–33% (for the conjugates). The content of β-structures changed from 15% (for the native enzyme) to 18% (for the conjugate). The obtained data open new opportunities for the synthesis of L-asparaginase preparations with improved biocatalytic properties.

Activity of the telomerase catalytic subunit hTERT (human Telomerase Reverse Transcriptase) can be regulated by alternative splicing of its mRNA. At the present time, the 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 to inhibit telomerase activity due to the activation of hTERT alternative splicing in normal activated human CD4+ and CD8+ Т 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+ Т cells with cisplatin caused the changes in proportion of hTERT splice variants and inhibition of telomerase activity.

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.

In this work, a recombinant chemotaxis CheW protein from Thermotoga petrophila RKU-1 (TpeCheW) and its mutant homologue (TpeCheW-mut) have been obtained. Despite the low homology with the CheW protein of the Escherichia coli bacteria, these proteins do not cause metabolic overload and are well expressed by E. coli laboratory strains. A wide range of features and parameters important for isolation of the TpeCheW-mut protein, such as stability over a wide range of temperatures and pH values, high level of expression, solubility, and the possibility of using simple low-stage purification schemes, including heat pretreatment, has been recognized. Possible directions of using this protein in the practice of scientific and applied research have been formulated and justified.

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.

Apoptotic endonuclease EndoG plays a key role in the alternative splicing of mRNA of human TERT telomerase catalytic subunit. The aim of this work was to test the ability of EndoG to induce alternative splicing of mRNA of other genes and in other organisms. To determine new mRNA splice-variants, EndoG overexpression was induced in human, mouse and rat CD4+-T-lymphocytes followed by sequencing of total RNA of these cells. Sequencing results showed that besides TERT, EndoG induced alternative splicing of deoxyribonuclease I (DNase I), caspase-2 (Casp-2) and BCL-x. The expression level of EndoG strongly correlated with mRNA splicing-variants of TERT, DNase I, Casp-2, and BCL-x in intact CD4+-T cells of healthy donors as well as different lines of mice and rats. EndoG overexpression induced down-regulation of fulllength mRNAs of TERT, DNase I, Casp-2, and BCL-x and up-regulation of their short-length mRNAs. Alternative splicing of studied mRNAs resulted in down-regulation of enzymatic activity of proteins in vitro and in vivo. The results of this work confirm the ability of endonuclease EndoG to induce alternative splicing of several mRNAs in human, mice and rats.