Barnase-barstar Specific Interaction Regulates Car-T Cells Cytotoxic Activity toward Malignancy.

The development of CAR-T specific therapy made a revolution in modern oncology. Despite the pronounced therapeutic effects, this novel approach displayed several crucial limitations caused by the complications in pharmacokinetics and pharmacodynamics controls. The presence of the several severe medical complications of CAR-T therapy initiated a set of attempts aimed to regulate their activity in vivo. We propose to apply the barnase-barstar system to control the cytotoxic antitumor activity of CAR-T cells. To menage the regulation targeting effect of the system we propose to use barstar-modified CAR-T cells together with barnase-based molecules. Barnase was fused with designed ankyrin repeat proteins (DARPins) specific to tumor antigens HER2 (human epidermal growth factor receptor 2) The application of the system demonstrates the pronounced regulatory effects of CAR-T targeting.

[Direct Molecular Fishing of Zinc-Dependent Protein Partners of Amyloid-beta 1-16 with the Taiwan (D7H) Mutation and Phosphorylated Ser8 Residue].

We previously showed that the metal-binding domain 1-16 of intact amyloid-beta (Aß) is involved in interactions with a number of proteins from the cytosolic fraction of SK-N-SH human neuroblastoma cells in a zinc-dependent manner only. It is known that hereditary mutations in the Aß metal-binding domain (Aß(1-16)), which accelerate the development of Alzheimer's disease and post-translational modifications of amino acid residues, can significantly affect the domain's structure in the presence of zinc ions. In this work, using the molecular fishing methodology for Aß(l-16) isoforms with the Taiwanese mutation (D7H) and a phosphorylated Ser8 residue, proteins from the cytosol of SK-N-SH cells were found that are able to form zinc-dependent non-covalent complexes with these domains. The partner proteins identified for these isoforms differed from those for intact Aß(1-16). In contrast, the Aß(1-16) isoform with the English mutation (H6R) and the Aß(1-16) isoform containing both an isomerized Asp7 residue and phosphorylated Ser8 residue did not interact with cytosolic proteins. The results are useful for developing methods for rational modulation of protein-protein interactions involving natural isoforms of beta-amyloid, and also indicate the possible role of beta-amyloid with phosphorylated Ser8 as a molecule involved in normal physiological processes.

Innate Immune Protein Tag7 Stimulates the Appearance of Cytotoxic NK Cells after Incubation with Lymphocytes.

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.

[Overexpression of microRNAs miR-9, -98, and -199 Correlates with the Downregulation of HK2 Expression in Colorectal Cancer].

Glycolysis activation is one of the main features of energy metabolism in cancer cells that is associated with the increase in glycolytic enzyme synthesis, primarily, hexokinases (HKs), in many types of tumors. Conversely, in colorectal cancer (CRC) the decrease in the expression of HK2 gene, which encodes one of the key rate-limiting enzyme of glycolysis, was revealed, thus, the study of the mechanisms of its inhibition in CRC is of particular interest. To search for potential microRNAs, inhibiting the expression of HK2 in CRC, we have performed the analysis of data from "The Cancer Genome Atlas" (TCGA) and five microRNA-mRNA target interaction databases (TargetScan, DIANA microT, mirSVR (miRanda), PicTar, and miRTarBase) using original CrossHub software. Seven microRNAs containing binding site on mRNA HK2, which expression is negatively correlated with HK2 expression, were selected for further analysis. The expression levels of these microRNAs and mRNA HK2 were estimated by quantitative PCR on a set of CRC samples. It has been shown, that the expression of three microRNAs (miR-9-5p, -98-5p, and -199-5p) was increased and correlated negatively with mRNA level of HK2 gene. Thus, downregulation of HK2 gene may be caused by its negative regulation through microRNAs miR-9-5p, -98-5p, and -199-5p.

Lymphocytes incubated in the presence of IL-2 lose the capacity for chemotaxis but acquire antitumor activity.

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.

Apoptotic tumor cell death under the influence of the cytotoxic complex Tag7-Hsp70 is induced by interaction with the TNFR1 receptor.

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.

[Structural and Physicochemical Characteristics of Conformationally Stable alpha-Helical Oligopeptides].

The analysis of conformationally stable (conformational conservative) tetrapeptides selected from protein structures deposited in PDBSelect data bank has been fulfilled. The subset contained 943 tetrapeptide amino acid sequences and there were merely five 3D protein segment representatives for each sequence. As a result, the conclusion has been drawn on the basis of DSSP annotation analysis that in the majority of cases (900 of 943) alpha-helical conformation is obvious. Different than alpha-helix, in particular, the left-handed polyproline II helical conformation was observed in 43 sequences. The physical and chemical properties of conformationally stable peptides taken from the appropriate sample were estimated by the average hydrophobicity/hydrophilicity of tetrapeptides. The results of calculations show that the "neutrality" towards hydrophobicity/hydrophilicity is representative of conformationally stable oligopeptides. It should be noted, that dispersion of hydrophobicity/hydrophilicity distribution is sufficiently lower than for the test subsets. Thus, the conformationally stable oligopeptides present a distinct group of local protein structures which are very close with respect to conformational and physicochemical properties. In accordance with our developed theory of specific long range interactions these peptides are the objects being quite useful for effective mutual molecular recognition.

[Classification of amino acids based on a comparative analysis of contacts in DNA-protein complexes and specific DNA-protein interactions].

The classification of amino acid residues based on the events of contact formation between distinct amino acid and selected nucleotides was constructed. Thus, the most integral properties, that characterize interactions in organization of DNA-protein complexes, were used. We applied the Voronoi-Delaunay tessellation to draw statistics of contacts and area of contacts for the set included 1937 DNA-protein complexes. Similarities of amino acid residues have been searched for based on the comparison of corresponded rows and matrixes of contacts and areas of contacts. Nine measures of distance were used for estimation of rows similarity degree. The procedure of clustering amino acids in groups included three hierarchical and two nonhierarchical methods. A total tree was built using nine techniques of estimating distance with three hierarchical clustering methods. It was shown that clustering centers in the main groups are always constant while other relationships between objects vary. Clustering of binary associations was found for the most amino acids. Major classes of up to six amino acids correspond to the certain local structures of the polypeptide chain in the context of amino acid composition. These data should be taken into account when designing DNA-protein ligands.

Cell death of L-929 cells induced by cytotoxic complex Tag7-Hsp70 is analogous to the death of the same cells induced by TNF-α.

The identification and studying the molecular bases of functioning of new cytotoxic agents finds an important implication in developing drugs for fighting with tumors. While investigating the cytotoxic action of protein complex Tag7-Hsp70 which was opened in our laboratory previously we found that Tag7-Hsp70 demonstrated the same specificity in regard to different tumor target cells as it was for classical cytokine TNF-α. L-929 cells and Jurkat cells appeared to be good targets representing up to 30% of dead cells within a population and HeLa cells--bad targets representing less than 5% of dead cells after 20 h of incubation with either of the cytotoxic agents. While investigating the action of either TNF-α or Tag7-Hsp70 on L-929 cells we detected two peaks of death: after 3 h and after 20 h. For both cytotoxic agents we observed the first, smaller (13-15%), peak to be eliminated after the addition of caspase inhibitor YVAD-CHO and the second, greater (25-30%), peak to become even bigger in presence of caspase inhibitor. Probably, protein complex Tag7-Hsp70 interacts like TNF-α with a receptor on the surface of tumor cells that results in triggering two alternative mechanisms of programmed cell death: apoptosis and necroptosis.

Nanoantibodies for detection and blocking of bioactivity of human vascular endothelial growth factor A(165).

Nanoantibodies (single-domain antibodies, nanobodies) derived from noncanonical single-chain immunoglobulins provide an attractive tool for in vitro and in vivo diagnostics as well as for development of targeted drugs for clinical use. Nanoantibodies against several clinically important targets have been developed and are actively investigated. However, no development of nanoantibodies against vascular endothelial growth factor VEGF-A(165) has been reported. We describe here the generation of nanoantibodies derived from single-chain Bactrian camel immunoglobulins directed against VEGF-A(165). We demonstrate that these nanoantibodies are suitable for enzyme-linked immunoassay to quantify human VEGF-A(165) as well as for blocking its activity. Our results provide a basis for diagnostic kit development for quantification of VEGF-A(165), which emerges as a biomarker useful in various pathological conditions. In addition, the nanoantibodies might be used for development of therapeutic molecules targeting VEGF-A(165)-dependent pathological neoangiogenesis.

[Functional properties of the WNT11 new isoform, expressed in colon carcinoma cell line HT29].

Colon carcinoma is a common type of neoplastic transformation. Mechanisms of its establishment and progression have been studying for several decades. Aberrant activation of the canonical Wnt signaling is frequently observed in colon carcinoma cells. Moreover, expression of the "noncanonical" Wnt ligands is also detected in this type of cancer. However, the implication of the noncanonical Wnt signaling in carcinogenesis and colorectal cancer (CRC) progression is still unclear. Here, to elucidate the characteristic features of the noncanonical Wnt signaling activation in CRC the expression of the "noncanonical" ligand hWnt11 has been studied. It was shown for the first time that expression of the hWnt11 in CRC is accompanied by the alternative splicing. The new hWnt11 isoform (hWnt11sp3) has been identified. Unlike to hWnt11, this isoform is not secreted and lacks the ability to inhibit the canonical Wnt signaling. Considering the canonical Wnt signaling inhibiting activity of hWnt11, different functional properties of the ligand and its isoform may reflect a special role of the alternative splicing in carcinogenesis and tumor progression. Thus, due to the difference in their functional properties an existence of several Wnt isoforms should be taken into account for the investigation of the role of Wnt ligands.

[Study of testicular cancer gene expression in samples of oral leukoplakia and squamous cell carcinoma of the mouth].

Cancer-testis (CT) antigens are normally expressed mostly in human germ cells, there is also an aberrant expression in some tumor cells. This expression profile makes them potential tumor growth biomarkers and a promising target for tumor immunotherapy. Specificity of CT genes expression in oral malignant and potentially malignant diseases, e.g. oral leukoplakia, is not yet studied. Data on CT genes expression profile in leukoplakia would allow developing new diagnostic methods with potential value for immunotherapy and prophylaxis of leukoplakia malignization. In our study we compared CT genes expression in normal oral mucosa, oral leukoplakia and oral squamous cell carcinoma. We are the first to describe CT genes expression in oral leukoplakia without dysplasia. This findings make impossible differential diagnosis of oral leukoplakia and squamous cell carcinoma on the basis of CT genes expression. The prognostic value of CT genes expression is still unclear, therefore the longitudinal studies are necessary.

[Nuclear beta-catenin localization is not sufficient for canonical Wnt signaling activation in human meianoma cell lines].

In most cases, advanced stages of melanoma are practically incurable due to high metastatic potential of tumor cells. Multiple observations support the idea that aberrations in Wnt signaling pathway play a significant role in melanoma development and progression. Canonical Wnt signaling activation results in stabilization and accumulation of the major effector molecule called beta-catenin. Mutations promoting beta-catenin stabilization and, thereby, activation of canonical Wnt signaling pathway are frequently found in different cancers, but rarely observed in melanomas. Nevertheless, beta-catenin nuclear and cytoplasmic accumulation is the feature of many human melanoma cell lines and original tumors. That is why, the aim of the investigation was to elucidate the relation between beta-catenin intracellular localization and activity status of Wnt signaling pathway in human melanoma cell lines. Ten human melanoma cell lines were characterized on the basis of the following parameters: canonical Wnt ligand expression, intracellular beta-catenin localization, and activity status of canonical Wnt signaling pathway. Here, it has been demonstrated that nuclear localization of beta-catenin does not always correspond to active status canonical Wnt signaling pathway. Moreover, in the majority of cell lines with nuclear beta-catenin canonical Wnt signaling can't be activated by exogenous expression of an appropriate ligand. Human melanoma cell lines differ in activity of canonical Wnt signaling pathway as well as in mechanisms of its regulation. Therefore, the pathway-targeted potential antineoplastic therapy requires the formation of a "molecular pattern of cancer" for localization of the defect in Wnt signaling cascade in the each case.

[Wars of cells with the use of nanoweapons].

Some mechanisms of the antitumor action of the protein Tag7 have been considered, and three scenarios of the manifestation of cytotoxic effects during the formation of its complex with other proteins have been considered.

[HLA-E molecule induction on the surface of tumor cells protects them from cytotoxic lymphocytes].

Modern immunotherapy has developed powerful tools for mounting antitumor response which nevertheless have had only limited success in clinic. Tumor cells use different mechanisms to escape from immune system. Thus, one of the reasons of unsuccessful immunotherapy might be induction of tolerance of tumor-specific cytotoxic lymphocytes by tumor cells. Previously we have demonstrated expression of HLA-E molecule by the cells of melanoma cell lines. In this paper we have studied HLA-E-dependent mechanism of melanoma cell escape from immune response.

"Shielding" of Cytokine Induction by the Periodontal Microbiome in Patients with Periodontitis Associated with Type 2 Diabetes Mellitus.

Periodontal diseases, especially those with polymicrobial etiology, are often associated with type 2 diabetes mellitus, proceeding more severely and affecting the course of diabetes mellitus. Recently, this feature has been associated with the ability of periodontopathogen microflora to cause not only a local infectious process in the oral cavity, but also to interact with the human immune system and induce various systemic effects. We investigated changes in the salivary cytokine profile of patients with chronic periodontitis, associated and not associated with type 2 diabetes mellitus. We observed a statistically significant decrease of MCP-1/CCL2, GM-CSF, IL-5, IL-6, and IFN-γ in the saliva of patients with chronic periodontitis associated with type 2 diabetes mellitus in comparison with patients with chronic periodontitis only. All of these cytokines are associated with macrophage activation. These data are an important contribution to the elucidation of the mechanism of periodontopathogens involvement in the manifestation of the systemic effects of type 2 diabetes.

[Transcription and mRNA splicing of the human lactoferrin gene controlled by the regulatory region of the bovine alphaS1 casein gene in the mammary gland of transgenic mice and in mouse embryonic stem cells].

The regulatory region of the bovine alphaS1 casein gene was used to obtain two genetic constructs for expression of human lactoferrin in the mammary gland of transgenic animals. Several transfected mouse embryonic stem cell (ESC) lines and primary transgenic mice were generated with these constructs. Recombinant lactoferrin was not detected in milk of transgenic mice by Western blotting. However, a recombinant transcript was found in RNAs isolated from mammary glands of transgenic females during lactation and from transfected ESC lines.

[Catalytic autoantibodies in autoimmune myocarditis: clinical and pathogenetic implications].

AIM: To evaluate pathogenetic and clinical significance of autoantibodies (AAB) with catalytic activity in the serum of patients with autoimmune myocarditis (AM). MATERIAL AND METHODS: The study was made on the sera from 99 patients with AM of different course: malignant, benign, myocardiosclerosis (MCS). In addition to standard immunological parameters, the study was made of serum levels of anticardiomyosine-antiCM (protabzymes) and anti-DNA (DNA-abzymes) of AAB. After obtaining anti-CM and anti-DNA IgG-AT, we determined non-specific and specific proteolytic activity of anti-CM. RESULTS: Maximal specific activity of protabzymes was seen in 73% patients with malignant AM, it correlated with blood levels of anti-CM AAB, DNA-abzymes activity was very high in 45% patients. In MCS proteolytic activity of autoAT was absent in 61% patients. In benign AM occurrence of protabzymes was confirmed in 35% cases. Elevated DNA-hydrolyzing activity of DNA-abzymes occurred in 13% cases. The activity had no significant correlation with serum titers of AB. In MCS proteolytic activity of AAB was absent in 61% cases, but high activity of anti-CM AAB was in 28%. The activity of DNA-abzymes in 44% ranged considerably which, in seropositive cases, detected significant correlation with serum titers of DNA-binding autoAT. CONCLUSION: Evaluation of catalytic activity of AAB may be considered as a criterial test assessing the stage, clinical variants and severity of AM. It also permits formulation of the disease prognosis and its possible outcomes.