The search for new cytotoxic agents capable of lysing tumor cells is an important task in the fight against cancer. Here we have shown that the HspBP1 protein, the chaperone of the heat shock protein Hsp70, is able to form a complex with the previously discovered peptide (17.1) of the innate immunity protein Tag7. Experiments using thermophoresis demonstrated that the affinity of the Tag7 protein peptide 17.1 to the HspBP1 molecule is 100 times higher than that of the full-sized Tag7 molecule. The addition of the 17.1-HspBP1 complex to tumor cells induces apoptosis and necroptosis in them. The results obtained in this work can be used to develop promising antitumor drugs.

To carry out antitumor activity against cells that have lost surface antigens, human lymphocytes must have a certain repertoire of surface proteins capable of contacting a tumor cell and inducing programmed cell death in it. In this work, we showed that activation of healthy donor cells by IL-2 cytokine within 6 days causes the appearance of FasL, CD25, and LFA-1 proteins on CD8+CD25+ T lymphocytes, and also converts the LFA-1 protein into an active form having a high affinity for its target, ICAM-1 integrin. The appearance of these proteins on the surface of this subpopulation of lymphocytes allows them to induce programmed cell death in HLA-negative tumor cells.

One of the basic features of immune system is the ability to sustain balance between activation and suppression of effector lymphocytes. In this process a key role belongs to the subpopulation of cells called regulatory T cells (Treg). Many cancer and autoimmune diseases are caused by malfunctions of Treg, and investigation of this subpopulation is important for development of new therapeutic approaches. In this study, we demonstrate that regulatory T cells can migrate along the concentration gradient of Tag7-Mts1 complex, and also they produce agents that induce blood cells migration.

Noncovalent interactions between DNA and proteins are vital for cells. DNA-protein crosslinking may occur due to various endogenous and exogenous factors, as well as chemotherapeutic agents, and often affects the performance of DNA-binding proteins. Oligodeoxyribonucleotides with a reactive group can serve as a tool to investigate the structure and action of DNA-binding proteins. Here, we report the crosslinking of MutS, which is a sensor protein of DNA mismatch repair (MMR) in Escherichia coli, via the protein’s cysteine. This was realized via DNA that harboured a 2′-deoxy-2′-[3-(2-pyridyldithio)propionamide] group as part of a nucleoside at a given position, and two fluorophores. The proposed DNA duplex consisted of complementary oligonucleotides with breaks in the top and bottom strands. This feature allowed us to introduce the reactive group at different positions and made it possible to synthesize only the central fragment of the DNA with a minimal length. The MutS-DNA conjugate was obtained by thiol-disulfide exchange with quantitative yield and separated from the unreacted DNA by size-exclusion chromatography. Fluorescence resonance energy transfer was used to show that MutS remained functionally active within the conjugate, since the protein was able to change its conformation and DNA conformation as well.

The mismatch repair system (MMR) ensures the stability of genetic information during DNA replication in almost all organisms. Mismatch repair is initiated after recognition of a non-canonical nucleotide pair by the MutS protein and the formation of a complex between MutS and MutL. Eukaryotic and most bacterial MutL homologs function as endonucleases that introduce a single-strand break in the daughter strand of the DNA, thus activating the repair process. However, many aspects of the functioning of this protein remain unknown. We studied the ATPase and DNA binding functions of the MutL protein from the pathogenic bacterium Neisseria gonorrhoeae (NgoMutL), which exhibits endonuclease activity. For the first time, the kinetic parameters of ATP hydrolysis by the full-length NgoMutL protein were determined. Its interactions with single- and double-stranded DNA fragments of various lengths were studied. NgoMutL was shown to be able to efficiently form complexes with DNA fragments that are longer than 40 nucleotides. Using modified DNA duplexes harboring a 2-pyridyldisulfide group on linkers of various lengths, we obtained NgoMutL conjugates with DNA for the first time. According to these results, the Cys residues of the wild-type protein are located at a distance of approximately 18–50 Å from the duplex. The efficiency of the affinity modification of Cys residues in NgoMutL with reactive DNAs was shown to decrease in the presence of ATP or its non-hydrolyzable analog, as well as ZnCl2, in the reaction mixture. We hypothesize that the conserved Cys residues of the C-terminal domain of NgoMutL, which are responsible for the coordination of metal ions in the active center of the protein, are involved in its interaction with DNA. This information may be useful in reconstruction of the main stages of MMR in prokaryotes that are different from γ-proteobacteria, as well as in the search for new targets for drugs against N. gonorrhoeae.

Nicking endonucleases (NEs) are a small, poorly studied family of restriction endonucleases. The enzymes recognize a target sequence in DNA, but catalyze the hydrolysis of only one strand. The mechanism of their action is important to study because NEs with new specificities are necessary to design to solve the practical tasks of biotechnology. One of the modern approaches for investigation of protein-nucleic acid interactions is fluorescence spectroscopy, which involves the introduction of fluorophores into proteins, mainly through Cys residues due to the high reactivity of their thiol group. To implement this approach, it is necessary to clarify the role of Cys residues in the functioning of the native protein and the possible consequences of their modification. Crosslinking was used to study whether Cys residues are close to DNA in the complex with NE BspD6I. Reactions were carried out using the wild-type enzyme, its mutant form NE BspD6I(C11S/C160S), and modified DNA duplexes containing the 2-pyridyldisulfide group at the C2' atom of the sugar-phosphate moiety in different positions of the oligonucleotide strand. The Cys residues of NE BspD6I were for the first time shown to be in close proximity to DNA during the binding process, including the step of a nonspecific complex formation. The substitutions C11S and C160S in the N-terminal domain of the enzyme slightly decreased the efficiency of substrate hydrolysis. Construction of a cysteine-free NE BspD6I variant and examination of its properties will provide additional information about the functional significance of the Cys residues for this unique enzyme.

Diketone DNA derivatives have been proposed to modify the guanidine group of Arg in proteins. The β-diketo group at the C2' atom of the sugar phosphate moiety has been introduced in DNA by acylation of oligonucleotide precursors, i.e., DNA fragments containing 2'-amino-2'-deoxyuridine, which have been synthesized by the chemical automatic synthesis. Water-soluble N-[3-(dimethylamino)propyl]-N′-ethylcarbodiimide (EDC) and 4,6-dioxoheptanoic acid have been used in the reaction. The ability of oligodeoxyribonucleotides containing the 2'-β-diketo group to react with guanidine, Nα-Boc-L-arginine, and Nα-Dns-L-arginine has been demonstrated. The introduction of this modification into one of the strands of the 15-base pair DNA duplex has been shown to lead to its destabilization. The conjugate formation of MutS and MutL proteins from the E. coli mismatch repair system with 17-base pair DNA duplexes containing the 2'-deoxy-2'-(4,6-dioxoheptylamido)uridine residue has been detected for the first time. To increase the selectivity of the DNA ligands containing the β-diketo group in the reaction with the Arg residues of proteins, we have proposed to treat the reaction mixture with hydroxylamine. This treatment leads to the cleavage of Schiff bases, which are formed with the involvement of lysine residues.

Tag7 (PGRP-S) is an innate immune protein that is involved in the antibacterial and antitumor defense and stimulates the maturation of cytotoxic lymphocyte subpopulations. It was found that the incubation of lymphocytes with Tag7 for 3 days promotes the appearance of cytotoxic NK cells that are active against a number of tumor cell lines.

Nicking endonucleases (NE) are a special group of the restriction endonucleases family. These unique enzymes catalyze the hydrolysis of only one DNA strand in a predetermined position relative to the recognition site. In this review, we summarize the engineering methods for NE construction: inactivation of the catalytic center of restriction endonucleases, disruption of enzyme dimerization interface, or random mutagenesis of the genes оf restriction endonucleases. The main methods of NE application in biotechnology and gene engineering are described. NE-mediated amplification for the enhancement of analytical signal in the detection of nucleic acids, proteins, and small molecules is characterized.

Tag7 (PGRP-S) – белок врождённого иммунитета, который принимает участие в антибактериальной и противоопухолевой защите, а также стимулирует созревание цитотоксических субпопуляций лимфоцитов. Обнаружили, что инкубация в течение 3 сут лимфоцитов с Tag7 способствует появлению цитотоксических NK-клеток, активных против ряда опухолевых клеточных линий.

The discovery of new chemokines that induce the migration of lymphocytes to the infection site is important for the targeted search for therapeutic agents in immunotherapy. We recently showed that Tag7 (PGLYRP1), an innate immunity protein, forms a stable complex with the Ca2+ -binding protein Mts1 (S100A4), which is able to induce lymphocyte movement, although the individual Tag7 and Mts1 do not have this activity. The purpose of this study is to identify receptors that induce the migration of lymphocytes along the concentration gradient of the Tag7-Mts1 complex, and the components of this complex capable of interacting with these receptors. The study investigated the migration of human PBMC under the action of the Tag7-Mts1complex. PBMC of healthy donors were isolated using a standard Ficoll-Hypaque gradient centrifugation procedure. It has been established that the movement of PBMC along the concentration gradient of the Tag7-Mts1 complex is induced by the classical chemotactic receptors CCR5 and CXCR3. It has been shown that only Mts1 is able to bind to the extracellular domain of CCR5, however, this binding is not enough to induce cell movement. A comparative analysis of the primary and 3D structures of the three proteins revealed the homology of the amino acid sequence fragments of the Tag7-Mts1 protein complex with different sites of the CCR5 receptor ligand - MIP1α protein. In conclusion, it should be noted that the Tag7-Mts1 complex can be considered as a new ligand of the classical chemotactic receptors CCR5 and CXCR3.

We have shown that in the human peripheral blood cells, the innate immunity protein Tag7 can activate a subpopulation of CD3+CD4+CD25+ cells, which have antitumor activity. These cells can induce lysis of HLA-negative tumor cell lines. The Hsp70 stress molecule on the surface of the tumor cells is used as a recognition target, while the Tag7 protein on the lymphocyte membrane acts as a receptor for Hsp70. We have also demonstrated that this subpopulation of the CD4+CD25+ cells is CD127 positive and hence is not the Treg cells. Our data suggest that this subpopulation of cells is identical to the CD4+CD25+ lymphocytes, which are activated in the leukocyte pool by the IL-2 cytokine.

Naïve non-activated lymphocytes are capable of releasing the chemoattractant complex Tag7–Mts1 and can migrate along the gradient of its concentration. After activation of these cells by IL-2, they acquire the abilities to kill tumor cells and to release the cytotoxic Tag7–Hsp70 complex, which is accompanied by a loss of both the Tag7–Mts1-mediated lymphocyte chemotaxis and the ability to release this chemoattractant into the conditioned medium.

The results of comparative analysis of interaction between the protein cytotoxic complex Tag 7–Hsp70 and the Tag 7 component of this complex with TNFR1 receptor in solution and in tumor cells are presented.