L-asparaginases (EC 3.5.1.1) are a family of enzymes that catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. These proteins with different biochemical, physicochemical and pharmacological properties are found in many organisms, including bacteria, fungi, algae, plants and mammals. To date, asparaginases from E. coli and Dickeya dadantii (formerly known as Erwinia chrysanthemi) are widely used in hematology for the treatment of lymphoblastic leukemias. However, their medical use is limited by side effects associated with the ability of these enzymes to hydrolyze L-glutamine, as well as the development of immune reactions. To solve these issues, gene-editing methods to introduce amino-acid substitutions of the enzyme are implemented. In this review, we focused on molecular analysis of the mechanism of enzyme action and to optimize the antitumor activity.

DNA nanotechnology has significantly advanced and might be used in biomedical applications, drug delivery, and cancer treatment during the past few decades. DNA nanomaterials are widely used in biomedical research involving biosensing, bioimaging, and drug delivery since they are remarkably addressable and biocompatible. Gradually, modified nucleic acids have begun to be employed to construct multifunctional DNA nanostructures with a variety of architectural designs. Aptamers are single-stranded nucleic acids (both DNAs and RNAs) capable of self-pairing to acquire secondary structure and of specifically binding with the target. Diagnosis and tumor therapy are prospective fields in which aptamers can be applied. Many DNA nanomaterials with three-dimensional structures have been studied as drug delivery systems for different anticancer medications or gene therapy agents. Different chemical alterations can be employed to construct a wide range of modified DNA nanostructures. Chemically altered DNA-based nanomaterials are useful for drug delivery because of their improved stability and inclusion of functional groups. In this work, the most common oligonucleotide nanomaterials were reviewed as modern drug delivery systems in tumor cells.

Alternative splicing (AS) of human telomerase catalytic subunit (hTERT, human telomerase reverse transcriptase) pre-mRNA strongly regulates telomerase activity. Several proteins can regulate AS in a cell type-specific manner and determine the functions of cells. In addition to being involved in telomerase activity regulation, AS provides cells with different splice variants that may have alternative biological activities. The modulation of telomerase activity through the induction of hTERT AS is involved in the development of different cancer types and embryos, and the differentiation of stem cells. Regulatory T cells may suppress the proliferation of target human and murine T and B lymphocytes and NK cells in a contact-independent manner involving activation of TERT AS. This review focuses on the mechanism of regulation of hTERT pre-mRNA AS and the involvement of splice variants in physiological and pathological processes.

Regulatory T cells (Tregs) play a key role in maintaining immune balance and regulating the loss of self-tolerance mechanisms in various autoimmune diseases, including primary Sjögren’s syndrome (pSS). With the development of pSS primarily in the exocrine glands, lymphocytic infiltration occurs in the early stages, mainly due to activated CD4⁺ T cells. Subsequently, in the absence of rational therapy, patients develop ectopic lymphoid structures and lymphomas. While the suppression of autoactivated CD4⁺ T cells is involved in the pathological process, the main role belongs to Tregs, making them a target for research and possible regenerative therapy. However, the available information about their role in the onset and progression of this disease seems unsystematized and, in certain aspects, controversial. In our review, we aimed to organize the data on the role of Tregs in the pathogenesis of pSS, as well as to discuss possible strategies of cell therapy for this disease. This review provides information on the differentiation, activation, and suppressive functions of Tregs and the role of the FoxP3 protein in these processes. It also highlights data on various subpopulations of Tregs in pSS, their proportion in the peripheral blood and minor salivary glands of patients as well as their role in the development of ectopic lymphoid structures. Our data emphasize the need for further research on Tregs and highlight their potential use as a cell-based therapy.

Genetic engineering for heterologous expression has advanced in recent years. Model systems such as Escherichia coli, Bacillus subtilis and Pichia pastoris are often used as host microorganisms for the enzymatic production of L-asparaginase, an enzyme widely used in the clinic for the treatment of leukemia and in bakeries for the reduction of acrylamide. Newly developed recombinant L-asparaginase (L-ASNase) may have a low affinity for asparagine, reduced catalytic activity, low stability, and increased glutaminase activity or immunogenicity. Some successful commercial preparations of L-ASNase are now available. Therefore, obtaining novel L-ASNases with improved properties suitable for food or clinical applications remains a challenge. The combination of rational design and/or directed evolution and heterologous expression has been used to create enzymes with desired characteristics. Computer design, combined with other methods, could make it possible to generate mutant libraries of novel L-ASNases without costly and time-consuming efforts. In this review, we summarize the strategies and approaches for obtaining and developing L-ASNase with improved properties.

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine and food industry. Drawbacks of current commercial L-ASNases stimulate the search for better-producing sources of the enzyme, and extremophiles are especially attractive in this view. In this study, a novel L-asparaginase originating from the hyperthermophilic archaeon Thermococcus sibiricus (TsA) was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 90 °C and pH 9.0 with a specific activity of 2164 U/mg towards L-asparagine. Kinetic parameters K_M and V_max for the enzyme are 2.8 mM and 1200 µM/min, respectively. TsA is stable in urea solutions 0–6 M and displays no significant changes of the activity in the presence of metal ions Ni²⁺, Cu²⁺, Mg²⁺, Zn²⁺ and Ca²⁺ and EDTA added in concentrations 1 and 10 mmol/L except for Fe³⁺. The enzyme retains 86% of its initial activity after 20 min incubation at 90 °C, which should be enough to reduce acrylamide formation in foods processed at elevated temperatures. TsA displays strong cytotoxic activity toward cancer cell lines K562, A549 and Sk-Br-3, while normal human fibroblasts WI-38 are almost unsensitive to it. The enzyme seems to be a promising candidate for further investigation and biotechnology application.