[The Role of the WGR Domain in the Functions of PARP1 and PARP2].

The PARP1 and PARP2 proteins are members of the poly(ADP-ribose) polymerase family involved in the regulation of DNA repair and replication, RNA processing, ribosome biogenesis, transcription, cell division, and cell death. PARP1 and PARP2 are promising targets for the development of anticancer drugs and can be used in the treatment of cardiovascular, neurodegenerative, and other disorders. The WGR domain has been shown to play a central role in the functioning of PARP1 and PARP2 proteins. This review considers the mechanisms of functioning of WGR domains in the PARP1 and PARP2 proteins, which have several similar and specialized properties. Understanding these processes is of great interest to fundamental science and can contribute to the development of more effective and selective inhibitors of PARP1 and PARP2.

Properties and Activity of Peptide Derivatives of ACE2 Cellular Receptor and Their Interaction with SARS-CoV-2 S Protein Receptor-Binding Domain.

The aim of this work was to design and characterize peptides based on the α-helices h1 and h2 of the ACE2 receptor, forming the interaction interface between the receptor-binding domain (RBD) of the SARS-CoV-2 S protein and the cellular ACE2 receptor. Monomeric and heterodimeric peptides connected by disulfide bonds at different positions were synthesized. Solubility, RBD-binding affinity, and peptide helicity were experimentally measured, and molecular dynamics simulation was performed in various solvents. It was established that the preservation of the helical conformation is a necessary condition for the binding of peptides to RBD. The peptides have a low degree of helicity and low affinity for RBD in water. Dimeric peptides have a higher degree of helicity than monomeric ones, probably due to the mutual influence of helices. The degree of helicity of the peptides in trifluoroethanol is the highest; however, for in vitro studies, the most suitable solvent is a water-ethanol mixture.

Epinephrine affects gene expression levels and has a complex effect on biofilm formation in M icrococcus luteus strain C01 isolated from human skin.

In this study, the effect of epinephrine on the biofilm formation of Micrococcus luteus C01 isolated from human skin was investigated in depth for the first time. This hormone has a complex effect on biofilms in various systems. In a system with polytetrafluoroethylene (PTFE) cubes, treatment with epinephrine at a physiological concentration of 4.9 × 10-9 M increased the total amount of 72-h biofilm biomass stained with crystal violet and increased the metabolic activity of biofilms, but at higher and lower concentrations, the treatment had no significant effect. On glass fiber filters, treatment with the hormone decreased the number of colony forming units (CFUs) and changed the aggregation but did not affect the metabolic activity of biofilm cells. In glass bottom plates examined by confocal microscopy, epinephrine notably inhibited the growth of biofilms. RNA-seq analysis and RT-PCR demonstrated reproducible upregulation of genes encoding Fe-S cluster assembly factors and cyanide detoxification sulfurtransferase, whereas genes encoding the co-chaperone GroES, the LysE superfamily of lysine exporters, short-chain alcohol dehydrogenase and the potential c-di-GMP phosphotransferase were downregulated. Our results suggest that epinephrine may stimulate matrix synthesis in M. luteus biofilms, thereby increasing the activity of NAD(H) oxidoreductases. Potential c-di-GMP pathway proteins are essential in these processes.

Poly(ADP-Ribosyl) Code Functions.

Poly(ADP-ribosyl)ation plays a key role in cellular metabolism. Covalent poly(ADP-ribosyl)ation affects the activity of the proteins engaged in DNA repair, chromatin structure regulation, gene expression, RNA processing, ribosome biogenesis, and protein translation. Non-covalent PAR-dependent interactions are involved in the various types of cellular response to stress and viral infection, such as inflammation, hormonal signaling, and the immune response. The review discusses how structurally different poly(ADP-ribose) (PAR) molecules composed of identical monomers can differentially participate in various cellular processes acting as the so-called "PAR code." The article describes the ability of PAR polymers to form functional biomolecular clusters through a phase-separation in response to various signals. This phase-separation contributes to rapid spatial segregation of biochemical processes and effective recruitment of the necessary components. The cellular PAR level is tightly controlled by a network of regulatory proteins: PAR code writers, readers, and erasers. Impaired PAR metabolism is associated with the development of pathological processes causing oncological, cardiovascular, and neurodegenerative diseases. Pharmacological correction of the PAR level may represent a new approach to the treatment of various diseases.

Histone Tails Promote PARP1-Dependent Structural Rearrangements in Nucleosomes.

PARP 1 alters the wrapping of nucleosomal DNA on the histone octamer, thereby modulating the accessibility of different genome sites to nuclear protein factors. Here, we show that non-structured histone tails are involved in the PARP1-induced structural rearrangements in nucleosomes, facilitate and stabilize them, but do not affect the enzymatic activity of PARP1.

Human secreted proteins SLURP-1 and SLURP-2 control the growth of epithelial cancer cells via interactions with nicotinic acetylcholine receptors.

BACKGROUND AND PURPOSE: Nicotinic acetylcholine receptors (nAChRs) are a promising target for development of new anticancer therapies. Here we have investigated the effects of the endogenous human proteins SLURP-1 and SLURP-2, antagonists of nAChRs, on human epithelial cancer cells. EXPERIMENTAL APPROACH: Growth of epithelial cancer cells (A431, SKBR3, MCF-7, A549, HT-29) exposed to SLURP-1, SLURP-2, mecamylamine, atropine, timolol and gefitinib was measured by the WST-1 test. Expression levels of SLURP-1, α7-nAChR and EGF receptors and their distribution in cancer cells were studied by confocal microscopy and flow cytometry. Secretion of endogenous SLURP-1 by A431 cells under treatment with recombinant SLURP-1 was analysed by Western-blotting. KEY RESULTS: SLURP-1 and SLURP-2 significantly inhibited growth of A431, SKBR3, MCF-7 and HT-29 cells at concentrations above 1 nM, to 40-70% of the control, in 24 h. Proliferation of A549 cells was inhibited only by SLURP-1. The anti-proliferative activity of SLURPs on A431 cells was associated with nAChRs, but not with ß-adrenoceptors or EGF receptors. Action of gefitinib and SLURPs was additive and resulted almost complete inhibition of A431 cell proliferation during 24 h. Exposure of A431 cells to recombinant SLURP-1 down-regulated α7-nAChR expression and induced secretion of endogenous SLURP-1 from intracellular depots, increasing its concentration in the extracellular media. CONCLUSIONS AND IMPLICATIONS: SLURPs inhibit growth of epithelial cancer cells in vitro and merit further investigation as potential agents for anticancer therapy. LINKED ARTICLES: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.

Complexes of Peptide Blockers with Kv1.6 Pore Domain: Molecular Modeling and Studies with KcsA-Kv1.6 Channel.

Potassium voltage-gated Kv1.6 channel, which is distributed primarily in neurons of central and peripheral nervous systems, is of significant physiological importance. To date, several high-affinity Kv1.6-channel blockers are known, but the lack of selective ones among them hampers the studies of tissue localization and functioning of Kv1.6 channels. Here we present an approach to advanced understanding of interactions of peptide toxin blockers with a Kv1.6 pore. It combines molecular modeling studies and an application of a new bioengineering system based on a KcsA-Kv1.6 hybrid channel for the quantitative fluorescent analysis of blocker-channel interactions. Using this system we demonstrate that peptide toxins agitoxin 2, kaliotoxin1 and OSK1 have similar high affinity to the extracellular vestibule of the K+-conducting pore of Kv1.6, hetlaxin is a low-affinity ligand, whereas margatoxin and scyllatoxin do not bind to Kv1.6 pore. Binding of toxins to Kv1.6 pore has considerable inverse dependence on the ionic strength. Model structures of KcsA-Kv1.6 and Kv1.6 complexes with agitoxin 2, kaliotoxin 1 and OSK1 were obtained using homology modeling and molecular dynamics simulation. Interaction interfaces, which are formed by 15-19 toxin residues and 10 channel residues, are described and compared. Specific sites of Kv1.6 pore recognition are identified for targeting of peptide blockers. Analysis of interactions between agitoxin 2 derivatives with point mutations (S7K, S11G, L19S, R31G) and KcsA-Kv1.6 confirms reliability of the calculated complex structure.

Towards universal approach for bacterial production of three-finger Ly6/uPAR proteins: Case study of cytotoxin I from cobra N. oxiana.

Cytotoxins or cardiotoxins is a group of polycationic toxins from cobra venom belonging to the 'three-finger' protein superfamily (Ly6/uPAR family) which includes small ß-structural proteins (60-90 residues) with high disulfide bond content (4-5 disulfides). Due to a high cytotoxic activity for cancer cells, cytotoxins are considered as potential anticancer agents. Development of the high-throughput production methods is required for the prospective applications of cytotoxins. Here, efficient approach for bacterial production of recombinant analogue of cytotoxin I from N. oxiana containing additional N-terminal Met-residue (rCTX1) was developed. rCTX1 was produced in the form of E. coli inclusion bodies. Refolding in optimized conditions provided ∼6 mg of correctly folded protein from 1 L of bacterial culture. Cytotoxicity of rCTX1 for C6 rat glioma cells was found to be similar to the activity of wild type CTX1. The milligram quantities of 13C,15N-labeled rCTX1 were obtained. NMR study confirmed the similarity of the spatial structures of recombinant and wild-type toxins. Additional Met residue does not perturb the overall structure of the three-finger core. The analysis of available data for different Ly6/uPAR proteins of snake and human origin revealed that efficiency of their folding in vitro is correlated with the number of proline residues in the third loop and the surface area of hydrophobic residues buried within the protein interior. The obtained data indicate that hydrophobic core is important for the folding of proteins with high disulfide bond content. Developed expression method opens new possibilities for structure-function studies of CTX1 and other related three-finger proteins.

Modeling of the Binding of Peptide Blockers to Voltage-Gated Potassium Channels: Approaches and Evidence.

Modeling of the structure of voltage-gated potassium (KV) channels bound to peptide blockers aims to identify the key amino acid residues dictating affinity and provide insights into the toxin-channel interface. Computational approaches open up possibilities for in silico rational design of selective blockers, new molecular tools to study the cellular distribution and functional roles of potassium channels. It is anticipated that optimized blockers will advance the development of drugs that reduce over activation of potassium channels and attenuate the associated malfunction. Starting with an overview of the recent advances in computational simulation strategies to predict the bound state orientations of peptide pore blockers relative to KV-channels, we go on to review algorithms for the analysis of intermolecular interactions, and then take a look at the results of their application.

Interaction of linear cationic peptides with phospholipid membranes and polymers of sialic acid.

Polysialic acid (PSA) is a natural anionic polymer typically occurring on the outer surface of cell membranes. PSA is involved in cell signaling and intermolecular interactions with proteins and peptides. The antimicrobial potential of peptides is usually evaluated in model membranes consisting of lipid bilayers but devoid of either PSA or its analogs. The goal of this work was to investigate the possible effect of PSA on the structure of melittin (Mlt) and latarcins Ltc1K, Ltc2a, and the activity of these peptides with respect to model membranes. These peptides are linear cationic ones derived from the venom of bee (Mlt) and spider (both latarcins). The length of each of the peptides is 26 amino acid residues, and they all have antimicrobial activity. However, they differ with respect to conformational mobility, hydrophobic characteristics, and overall charge. In this work, using circular dichroism spectroscopy, we show that the peptides adopt an α-helical conformation upon interaction with either PSA or phospholipid liposomes formed of either zwitterionic or anionic phospholipids or their mixtures. The extent of helicity depends on the amino acid sequence and properties of the medium. Based on small angle X-ray scattering data and the analysis of the fluorescence spectrum of the Trp residue in Mlt, we conclude that the peptide forms an oligomeric complex consisting of α-helical Mlt and several PSA molecules. Both latarcins, unlike Mlt, the most hydrophobic of the peptides, interact weakly with zwitterionic liposomes. However, they bind anionic liposomes or those composed of anionic/zwitterionic lipid mixtures. Latarcin Ltc1K forms associates on liposomes composed of zwitterionic/anionic lipid mixture. The structure of the peptide associates is either disordered or of ß-sheet conformation. In all other cases the studied peptides adopt predominately α-helical conformation. In addition, we demonstrate that PSA inhibits membranolytic activity of Mlt and latarcin Ltc1K. These data suggest that the peptides, due to their high conformational lability, can vary structural and amphiphilic properties in the presence of PSA. As a result, various scenarios of the interaction of the peptides with membranes, whose surface is abundant with anionic polysaccharides, can take place. This can account for difficulties in understanding the structure-functional relationships in interactions of linear cationic peptides with biological membranes.

[application of the analytical transmission electron microscopy techniques for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in mammalian cells].

This work represents the results of the study on applicability of the modern methods of analytical transmission electron microscopy for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in A549 cell, human lung adenocarcinoma cell line. A comparative analysis of images of the nanoparticles in the cells obtained in the bright field mode of transmission electron microscopy, under dark-field scanning transmission electron microscopy and high-angle annular dark field scanning transmission electron was performed. For identification of nanoparticles in the cells the analytical techniques, energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy, were compared when used in the mode of obtaining energy spectrum from different particles and element mapping. It was shown that the method for electron tomography is applicable to confirm that nanoparticles are localized in the sample but not coated by contamination. The possibilities and fields of utilizing different techniques for analytical transmission electron microscopy for detection, visualization and identification of nanoparticles in the biological samples are discussed.

Receptor-binding domain of ephrin-A1: production in bacterial expression system and activity.

Eph receptor tyrosine kinases and their ligands, the ephrins, perform an important regulatory function in tissue organization, as well as participate in malignant transformation of cells. Ephrin-A1, a ligand of A class Eph receptors, is a modulator of tumor growth and progression, and the mechanism of its action needs detailed investigation. Here we report on the development of a system for bacterial expression of an ephrin-A1 receptor-binding domain (eA1), a procedure for its purification, and its renaturation with final yield of 50 mg/liter of culture. Functional activity of eA1 was confirmed by immunoblotting, laser scanning confocal microscopy, and flow cytometry. It is shown that monomeric non-glycosylated receptor-binding domain of ephrin-A1 is able to activate cellular EphA2 receptors, stimulating their phosphorylation. Ligand eA1 can be used to study the features of ephrin-A1 interactions with different A class Eph receptors. The created expression cassette is suitable for the development of ligands with increased activity and selectivity and experimental systems for the delivery of cytotoxins into tumor cells that overexpress EphA2 or other class A Eph receptors.

Multi-walled Сarbon Nanotubes Penetrate into Plant Cells and Affect the Growth of Onobrychis arenaria Seedlings.

Engineered nanoparticles (ENPs) are now being used in many sectors of industry; however, the impact of ENPs on the environment still requires further study, since their use, recycling, and accidental spill can result in the accumulation of nanoparticles in the atmosphere, soil, and water. Plants are an integral part of ecosystems; hence their interaction with ENPs is inevitable. It is important to understand the consequences of this interaction and assess its potential effects. The present research is focused on studying the effects of the industrial material Taunit, containing multi-walled carbon nanotubes (MWNTs), on plants, and testing of its ability to penetrate into plant cells and tissues. Taunit has been found to stimulate the growth of roots and stems and cause an increase in peroxidase activity inOnobrychis arenariaseedlings. Peroxidase activity increases with decreasing concentration of Taunit from 1,000 to 100 mg/l. MWNTs from Taunit were detected in the cells and tissues of seedling roots and leaves, implying the ability of MWNTs to penetrate into roots and accumulate there, as well as their ability to be transported into seedling leaves. Thus, the changes in the physiological parameters of plants are associated not only with MWNT adsorption on the root surface, as previously believed, but also with their penetration, uptake and accumulation in the plant cells and tissues.

N-terminal moiety of Antimicrobial peptide Ltc1-k increases its toxicity for eukaryotic cells.

The antimicrobial peptide Ltc1-K and its derivates without one, two, then three N-terminal amino acid residues were studied based on the hypothesis (backed by some experimental data) that the hydrophobic N-terminal moiety of linear cationic antimicrobial peptides defines their haemolytic activity. It was discovered that the excision of three N-terminal amino acid residues considerably decreases the peptide's toxicity for eukaryotic cells and simultaneously increases the selectivity of antibacterial activity for some bacteria species. Studies performed with the model membrane systems and human erythrocytes revealed that the main reason for the observed effect is a multifold decrease in the peptide's affinity to an eukaryotic cellular membrane enriched with zwitterionic phospholipids.

[Interaction of the photosensitizer 13,15-N-(3'-hydroxypropyl)cycloimide of chlorin p(6) with normal and cancerous blood cells].

The interaction of 13,15-N-(3'-hydroxypropyl)cycloimide of chlorin p(6) (CIC) with normal blood cells and human K562 and HL60 myeloid leukemia cells was studied. CIC was found to be bound by the erythrocyte membrane but did not penetrate into the cytoplasm. It is characterized by a diffuse distribution in the cytoplasm of normal leukocytes, whereas its diffuse distribution in K562 and HL60 cells is accompanied by perinuclear accumulation and binding to the plasma membrane. The average cytoplasmic concentration corresponding to the CIC accumulation in leukemic cells at saturation is 2.2 to 2.6 times higher than that in normal leukocytes. CIC is more intensely accumulated in granulocytes than in lymphocytes. The kinetics of the cellular uptake and efflux was characterized. The normal leukocytes and erythrocytes were found to be 1.5 times and 3 to 4 times less sensitive, respectively, to the photodynamic action of CIC than the K562 and HL60 cells.

[Effect of substituents on photochemical and biological properties of 13,15-N-cycloimide derivatives of chlorin p6].

The effect of electron-accepting substituents in position 3 of the chlorine p6 macrocycle in neutral and carboxyl-containing negatively charged cycloimide derivatives of chlorin p6 (CIC) on the photochemical and biological properties of these photosensitizers was studied. A relationship between the structure and properties of CICs was analyzed on the basis of information on their photoinduced cytotoxicity, efficiency of the generation of reactive oxygen species, photostability, intracellular localization, quantitative parameters of accumulation in cells, and cellular pharmacokinetics. It was shown that these compounds can be used for the development of photosensitizers with intense light absorption at 740 nm, controlled intracellular localization, and a high photodynamic activity toward tumor cells.

Comparative analysis of proapoptotic activity of cytochrome c mutants in living cells.

A non-traumatic electroporation procedure was developed to load exogenous cytochrome c into the cytoplasm and to study the apoptotic effect of cytochrome c, its K72-substitued mutants and "yeast --> horse" hybrid cytochrome c in living WEHI-3 cells. The minimum apoptosis-activating intracellular concentration of horse heart cytochrome c was estimated to be 2.7 +/- 0.5 microM (47 +/- 9 fg/cell). The equieffective concentrations of the K72A-, K72E- and K72L-substituted mutants of cytochrome c were five-, 15- and 70-fold higher. The "yeast --> horse" hybrid created by introducing S2D, K4E, A7K, T8K, and K11V substitutions (horse protein numbering) and deleting five N-terminal residues in yeast cytochrome c did not evoke apoptotic activity in mammalian cells. The apoptotic function of cytochrome c was abolished by the K72W substitution. The K72W-substituted cytochrome c possesses reduced affinity to the apoptotic protease activating factor-1 (Apaf-1) and forms an inactive complex. This mutant is competent as a respiratory-chain electron carrier and well suited for knock-in studies of cytochrome c-mediated apoptosis.

Comparative study of structure and activity of cytotoxins from venom of the cobras Naja oxiana, Naja kaouthia, and Naja haje.

Cytotoxins are positively charged polypeptides that constitute about 60% of all proteins in cobra venom; they have a wide spectrum of biological activities. By CD spectroscopy, cytotoxins CT1 and CT2 Naja oxiana, CT3 Naja kaouthia, and CT1 and CT2 Naja haje were shown to have similar secondary structure in an aqueous environment, with dominating beta-sheet structure, and to vary in the twisting angle of the beta-sheet and the conformation of disulfide groups. Using dodecylphosphocholine micelles and liposomes, CT1 and CT2 Naja oxiana were shown to incorporate into lipid structures without changes in the secondary structure of the peptides. The binding of CT1 and CT2 Naja oxiana with liposomes was associated with an increase in the beta-sheet twisting and a sign change of the dihedral angle of one disulfide group. The cytotoxins were considerably different in cytotoxicity and cooperativity of the effect on human promyelocytic leukemia cells HL60, mouse myelomonocytic cells WEHI-3, and human erythroleukemic cells K562. The most toxic CT2 Naja oxiana and CT3 Naja kaouthia possessed low cooperativity of interaction (Hill coefficient h = 0.6-0.8), unlike 10-20-fold less toxic CT1 and CT2 Naja haje (h = 1.2-1.7). CT1 Naja oxiana has an intermediate position on the cytotoxicity scale and is characterized by h = 0.5-0.8. The cytotoxins under study induced necrosis of HL60 cells and failed to activate apoptosis. The differences in cytotoxicity are supposed to be related not with features of the secondary structure of the peptides, but with interactions of side chains of variable amino acid residues with lipids and/or membrane proteins.

[Photobiological properties of 13,15-N-(carboxymethyl)- and 13,15-N-(2-carboxyethyl)cycloimide derivatives of chlorin p6]. / Fotobiologicheskie svoistva 13,15-N-(karboksimetil)- i 13,15-N-(2-karboksiétil)tsikloimidnykh proizvodnykh khlorina p6.

Lipophilic derivatives of chlorin p6, 13,15-N-(carboxymethyl)cycloimide methyl ester (CIC1) and 13,15-N-(2-carboxyethyl)cycloimide methyl ester (CIC2), were shown to absorb light in 710 nm region and to be efficient IR photosensitizers. They exhibit similar phototoxicities on the cells of A549 human lung adenocarcinoma, which are 40- and 100-fold higher than those of chlorin p6 and the clinically used Photogem, respectively, and are not toxic in the absence of light irradiation. The confocal spectral imaging technique allowed us to demonstrate that the high phototoxicity of CIC1 and CIC2 is due to their ability to readily penetrate to cells and to be bound to the cell membranes and lipid-containing structures in the monomeric photoactive form. Under the irradiation, the membrane-bound CIC1 and CIC2 are characterized by high quantum yields of singlet oxygen generation (0.6 and 0.65, respectively) and the inability to produce hydroxyl radicals. A 1.5-microM content of CIC1 and CIC2 in the incubation medium provides for their average cytoplasmic concentrations of 21 and 16.5 microM, respectively. The incubation times to achieve 50% level of maximum accumulation for CIC1 and CIC2 in A549 cells are 30 +/- 6 and 24 +/- 12 min, and the times for 50% release of the dyes from the cells are 17 +/- 4 and 50 +/- 10 min, respectively. A diffuse distribution with the predominant accumulation in the membranes of the Golgi apparatus and mitochondria is characteristic of both CIC2 and CIC1, whereas, in addition, CIC1 is considerably accumulated in lipid droplets (cellular organelles responsible for the storage and metabolism of neutral lipids and steryl esters). Our results demonstrate that changes in the structure of the imide substituent could affect the intracellular localization and the rate of release of chlorin p6 cycloimide derivatives from cells while preserving their high photodynamic activity.