Alexey M. Nesterenko

Noggin4 is a long-range inhibitor of Wnt8 signalling that regulates head development in Xenopus laevis

Noggin4 is a Noggin family secreted protein whose molecular and physiological functions remain unknown. In this study, we demonstrate that in contrast to other Noggins, Xenopus laevis Noggin4 cannot antagonise BMP signalling; instead, it specifically binds to Wnt8 and inhibits the Wnt/β -catenin pathway. Live imaging demonstrated that Noggin4 diffusivity in embryonic tissues significantly exceeded that of other Noggins. Using the Fluorescence Recovery After Photobleaching (FRAP) assay and mathematical modelling, we directly estimated the affinity of Noggin4 for Wnt8 in living embryos and determined that Noggin4 fine-tune the Wnt8 posterior-to-anterior gradient. Our results suggest a role for Noggin4 as a unique, freely diffusing, long-range inhibitor of canonical Wnt signalling, thus explaining its ability to promote head development.

Molecular-dynamic simulation of phospholipid bilayers: Ion distribution at the surface of neutral and charged bilayer in the liquid crystalline state

The electric field and ion distribution at the surface of neutral and charged lipid bilayers (BeCl2 and dipalmitoyl phosphatidylcholine/dipalmitoyl phosphatidylserine (DPPC/DPPS) + KCl) were studied with molecular dynamic (MD) methods. It is shown that the contributions of lipid molecules, water and ions to the electric potential compensate each other in the region of the diffuse double layer and decrease the potential value close to zero. It is also demonstrated that the ion distribution at the charged surface is determined not only by the electrostatic ion-medium interaction. The total energy of this interaction was compared with the potential of mean ion force. It was shown that cations and anions have a different effect on the state of water molecules at the surface. The order parameter of water in the system DPPC + BeCl2 and the Clion distribution have the extremum at the distance of 10 α atoms of the phospholipid glycerol. This position was chosen as the “electrical” interface of the electrical double layer (EDL) for all lipid systems studied. The potential of mean force of counter ions in EDL allows us to obtain the value of potential at the lipid surface suitable for experimental test of the MD data. This surface potential and surface charge density was found from MD simulation different electrolyte concentrations and DPPS content of 20, 40 and 60% in the mixture with DPPC and was shown to be in a good agreement with the Gouy-Chapman-Stern model upon fitting parameters close to their experimental values.

Atomic-Scale Molecular Dynamics Simulations of DNA-Polycation Complexes: Two Distinct Binding Patterns.

Synthetic cationic polymers represent a promising class of delivery vectors for gene therapy. Here, we employ atomistic molecular dynamics simulations to gain insight into the structure and properties of complexes of DNA with four linear polycations: polyethylenimine (PEI), poly-l-lysine (PLL), polyvinylamine (PVA), and polyallylamine (PAA). These polycations differ in their polymer geometries, protonation states, and hydrophobicities of their backbone chains. Overall, our results demonstrate for the first time the existence of two distinct patterns of binding of DNA with polycations. For PEI, PLL, and PAA, the complex is stabilized by the electrostatic attraction between protonated amine groups of the polycation and phosphate groups of DNA. In contrast, PVA demonstrates an alternative binding pattern as it gets embedded into the DNA major groove. It is likely that both the polymer topology and affinity of the backbone chain of PVA to the DNA groove are responsible for such behavior. The differences in binding patterns can have important biomedical implications: embedding PVA into a DNA groove makes it less sensitive to changes in the aqueous environment (pH level, ionic strength, etc.) and could therefore hinder the intracellular release of genetic material from a delivery vector, leading to lower transfection activity.

Affinity of the heparin binding motif of Noggin1 to heparan sulfate and its visualization in the embryonic tissues

Heparin binding motifs were found in many secreted proteins and it was suggested that they are responsible for retardation of the protein diffusion within the intercellular space due to the binding to heparan sulfate proteoglycanes (HSPG). Here we used synthetic FITC labeled heparin binding motif (HBM peptide) of the Xenopus laevis secreted BMP inhibitor Noggin1 to study its diffusion along the surface of the heparin beads by FRAP method. As a result, we have found out that diffusivity of HBM-labeled FITC was indeed much lesser than those predicted by theoretical calculations even for whole protein of the Noggin size. We also compared by isothermal titration calorimetry the binding affinity of HBM and the control oligolysine peptide to several natural polyanions including heparan sulfate (HS), heparin, the bacterial dextran sulfate and salmon sperm DNA, and demonstrated that HBM significantly exceeds oligolysine peptide in the affinity to HS, heparin and DNA. By contrast, oligolysine peptide bound with higher affinity to dextran sulfate. We speculate that such a difference may ensure specificity of the morphogen binding to HSPG and could be explained by steric constrains imposed by different distribution of the negative charges along a given polymeric molecule. Finally, by using EGFP-HBM recombinant protein we have visualized the natural pattern of the Noggin1 binding sites within the X. laevis gastrula and demonstrated that these sites forms a dorsal-ventral concentration gradient, with a maximum in the dorsal blastopore lip. In sum, our data provide a quantitative basis for modeling the process of Noggin1 diffusion in embryonic tissues, considering its interaction with HSPG.

Conformational and phase transitions in DNA—photosensitive surfactant solutions: Experiment and modeling

DNA binding to trans‐ and cis‐isomers of azobenzene containing cationic surfactant in 5 mM NaCl solution was investigated by the methods of dynamic light scattering (DLS), low‐gradient viscometry (LGV), atomic force microscopy (AFM), circular dichroism (CD), gel electrophoresis (GE), flow birefringence (FB), UV–Vis spectrophotometry. Light‐responsive conformational transitions of DNA in complex with photosensitive surfactant, changes in DNA optical anisotropy and persistent length, phase transition of DNA into nanoparticles induced by high surfactant concentration, as well as transformation of surfactant conformation under its binding to macromolecule were studied. Computer simulations of micelles formation for cis‐ and trans‐isomers of azobenzene containing surfactant, as well as DNA‐surfactant interaction, were carried out. Phase diagram for DNA‐surfactant solutions was designed. The possibility to reverse the DNA packaging induced by surfactant binding with the dilution and light irradiation was shown.

Chapter Six – Interaction of Polylysines with the Surface of Lipid Membranes: The Electrostatic and Structural Aspects

Abstract The topic correlates electrostatic effects induced by polylysine (PL) adsorption at the lipid membrane surface with data of alternative methods sensitive to lipid bilayer structure. Comparison of electrokinetic data for liposomes from anionic lipids (cardiolipin, phosphatidylserine) and results of boundary potential (BP) measurements with lipid membranes shows effects in two opposite directions: fast positive changes of BP due to adsorption of polycations at the outer membrane surface and slow negative changes that can be attributed to alteration of the dipole component of BP. The latter effect does not depend on the polymer length and may be caused by lipid interaction with lysine as a basic unit of these polypeptides. Molecular dynamic simulation points out the possible mechanism of the dipole effect, which could be caused by reduced number of H-bonds to PO 4 groups upon the lysine adsorption. Atomic force microscopy visualized the geometry of clusters formed by PL of different lengths at the lipid bilayer. Isotherm titration calorimetry and the technique of lipid monolayers reveal the similarity in polypeptide and inorganic multivalent cation effects on the lateral lipid condensation accompanied by dipole effects.The topic correlates electrostatic effects induced by polylysine (PL) adsorption at the lipid membrane surface with data of alternative methods sensitive to lipid bilayer structure. Comparison of electrokinetic data for liposomes from anionic lipids (cardiolipin, phosphatidylserine) and results of boundary potential (BP) measurements with lipid membranes shows effects in two opposite directions: fast positive changes of BP due to adsorption of polycations at the outer membrane surface and slow negative changes that can be attributed to alteration of the dipole component of BP. The latter effect does not depend on the polymer length and may be caused by lipid interaction with lysine as a basic unit of these polypeptides. Molecular dynamic simulation points out the possible mechanism of the dipole effect, which could be caused by reduced number of H-bonds to PO4 groups upon the lysine adsorption. Atomic force microscopy visualized the geometry of clusters formed by PL of different lengths at the lipid bilayer. Isotherm titration calorimetry and the technique of lipid monolayers reveal the similarity in polypeptide and inorganic multivalent cation effects on the lateral lipid condensation accompanied by dipole effects.

Molecular-dynamic simulation of DPPC bilayer in different phase state: Hydration and electric field distribution in the presence of Be2+ cations

New molecular-dynamic topology of phosphatidylcholine bilayer (DPPC) in total atomic OPLS force field was developed and used to study the structural characteristics of liquid-crystalline and gel state of lipid bilayer in the absence and in the presence of Na+ and Be2+ cations adsorbed at the interface and different in their affinity. The parameters of bilayer geometry, the amount of surface water, and the electrostatic potential distribution were estimated quantitatively from the simulation in both phase states. The azimuthal angle of hydrocarbon chains was found nearly the same in the region of each monolayer in gel state. The amount of surface water decreases upon bilayer “freezing” mainly by loss of water molecules not participating in H-bonds between lipid headgroups. The cation adsorption was shown to have a small effect on these H-bonded water molecules, whereas Be2+ appeared to retain surface water in the bilayer upon its freezing. The electric potential distribution in the normal direction to the membrane-water interface had a similar shape in any bilayer phase state regardless of the presence of the adsorbed cations. Analysis of the microscopic nature of the electric potential revealed a mutual compensation of the contributions of the main structural components of the system (lipids, water, and ions). The boundary potential increased by 116 mV for pure DPPC, by 212 mV in the presence of Na+, and by 133 mV in the presence of Be2+ upon the phase transition of bilayer to the gel state. The boundary potential difference in the presence of Na+ and Be2+ and its change at the bilayer phase transition are in a good agreement with the experimental data published earlier [Ermakov Yu.A., 1993].

The interaction of secreted proteins Noggin4 and Wnt8 from Xenopus laevis embryos

We demonstrated that the secreted protein Noggin4 from Xenopus laevis was capable of the in vitro binding to the secreted factor Wnt8, one of the ligands of the Wnt/betaCatenin signaling pathway. It was also shown that posttranslational modifications occurring during secretion of these proteins from the embryonic cells were necessary for their effective interaction. Also, we proposed a method for the preparation of physiologically active secreted morphogenic proteins from the intercellular space of the Xenopus laevis embryos.

Conformational Dynamics of the Single Lipopolysaccharide O-Antigen in Solution.

The O-antigen is the most variable and highly immunogenic part of the lipopolysaccharide molecule that covers the surface of Gram-negative bacteria and makes up the first line of cellular defense. To provide insight into the details of the O-antigen arrangement on the membrane surface, we simulated its behavior in solution by molecular dynamics. We developed the energetically favorable O-antigen conformation by analyzing free-energy distributions for its disaccharide fragments. Starting from this conformation, we simulated the behavior of the O-antigen chain on long timescales. Depending on the force field and temperature, the single molecule can undergo reversible or irreversible coil-to-globule transitions. The mechanism of these transitions is related either to the rotation of the carbohydrate residues around O-glycosidic bonds or to flips of the pyranose rings. We found that the presence of rhamnose in the O-antigen chain crucially increases its conformational mobility.

Morphogene adsorption as a Turing instability regulator: Theoretical analysis and possible applications in multicellular embryonic systems.

The Turing instability in the reaction-diffusion system is a widely recognized mechanism of the morphogen gradient self-organization during the embryonic development. One of the essential conditions for such self-organization is sharp difference in the diffusion rates of the reacting substances (morphogens). In classical models this condition is satisfied only for significantly different values of diffusion coefficients which cannot hold for morphogens of similar molecular size. One of the most realistic explanations of the difference in diffusion rate is the difference between adsorption of morphogens to the extracellular matrix (ECM). Basing on this assumption we develop a novel mathematical model and demonstrate its effectiveness in describing several well-known examples of biological patterning. Our model consisting of three reaction-diffusion equations has the Turing-type instability and includes two elements with equal diffusivity and immobile binding sites as the third reaction substance. The model is an extension of the classical Gierer-Meinhardt two-components model and can be reduced to it under certain conditions. Incorporation of ECM in the model system allows us to validate the model for available experimental parameters. According to our model introduction of binding sites gradient, which is frequently observed in embryonic tissues, allows one to generate more types of different spatial patterns than can be obtained with two-components models. Thus, besides providing an essential condition for the Turing instability for the system of morphogen with close values of the diffusion coefficients, the morphogen adsorption on ECM may be important as a factor that increases the variability of self-organizing structures.