Small‐Molecule Inhibitors of AF6 PDZ‐Mediated Protein–Protein Interactions

PDZ (PSD‐95, Dlg, ZO‐1) domains are ubiquitous interaction modules that are involved in many cellular signal transduction pathways. Interference with PDZ‐mediated protein–protein interactions has important implications in disease‐related signaling processes. For this reason, PDZ domains have gained attention as potential targets for inhibitor design and, in the long run, drug development. Herein we report the development of small molecules to probe the function of the PDZ domain from human AF6 (ALL1‐fused gene from chromosome 6), which is an essential component of cell–cell junctions. These compounds bind to AF6 PDZ with substantially higher affinity than the peptide (Ile‐Gln‐Ser‐Val‐Glu‐Val) derived from its natural ligand, EphB2. In intact cells, the compounds inhibit the AF6–Bcr interaction and interfere with epidermal growth factor (EGF)‐dependent signaling.     

Characterization of a putative phosphorylation switch: adaptation of SPOT synthesis to analyze PDZ domain regulation mechanisms

Transient macromolecular complexes are often formed by protein-protein interaction domains (e.g., PDZ, SH2, SH3, WW), which are often regulated (positively or negatively) by phosphorylation. To address the in vitro analysis of PDZ domain regulation by such phosphorylation, we improved the inverted peptide method. This method is based on standard SPOT synthesis, followed by inversion of the peptide under acidic conditions to generate the free C termini necessary for PDZ domain ligand recognition. The benefit of the newly introduced acidic conditions is the preservation of the incorporated phosphate group during peptide synthesis. Furthermore, the improved method is more robust and shows an increased signal-to-noise ratio. As representative examples, we used the AF6, ERBIN, and SNA1 (alpha-1-syntrophin) PDZ domains to analyze the influence of ligand-position-dependent phosphorylation. We could clearly demonstrate severe down-regulation by phosphorylation of the PDZ ligand position -2 (<50 %) and slightly less at position -1 ( approximately 50 %). These results are specific and reproducible for all three PDZ domains. Finally, we confirmed the influence of negative regulation by using the protein kinase BCR as the AF6 PDZ domain ligand. For the first time, this approach allows the SPOT synthesis technique to be used to screen large libraries of phosphorylated peptides in vitro. This should ultimately help in the identification of phosphorylation-dependent regulation mechanisms in vivo.     

Structure and Stability of Carbohydrate–Lipid Interactions. Methylmannose Polysaccharide–Fatty Acid Complexes

We report a detailed study of the structure and stability of carbohydrate–lipid interactions. Complexes of a methylmannose polysaccharide (MMP) derivative and fatty acids (FAs) served as model systems. The dependence of solution affinities and gas‐phase dissociation activation energies (E_a) on FA length indicates a dominant role of carbohydrate–lipid interactions in stabilizing (MMP+FA) complexes. Solution ¹H NMR results reveal weak interactions between MMP methyl groups and FA acyl chain; MD simulations suggest the complexes are disordered. The contribution of FA methylene groups to the E_a is similar to that of heats of transfer of n‐alkanes from the gas phase to polar solvents, thus suggesting that MMP binds lipids through dipole‐induced dipole interactions. The MD results point to hydrophobic interactions and H‐bonds with the FA carboxyl group. Comparison of collision cross sections of deprotonated (MMP+FA) ions with MD structures suggests that the gaseous complexes are disordered.     

Syndecan Transmembrane Domain Specifically Regulates Downstream Signaling Events of the Transmembrane Receptor Cytoplasmic Domain

Despite the known importance of the transmembrane domain (TMD) of syndecan receptors in cell adhesion and signaling, the molecular basis for syndecan TMD function remains unknown. Using in vivo invertebrate models, we found that mammalian syndecan-2 rescued both the guidance defects in C. elegans hermaphrodite-specific neurons and the impaired development of the midline axons of Drosophila caused by the loss of endogenous syndecan. These compensatory effects, however, were reduced significantly when syndecan-2 dimerization-defective TMD mutants were introduced. To further investigate the role of the TMD, we generated a chimera, 2eTPC, comprising the TMD of syndecan-2 linked to the cytoplasmic domain of platelet-derived growth factor receptor (PDGFR). This chimera exhibited SDS-resistant dimer formation that was lost in the corresponding dimerization-defective syndecan-2 TMD mutant, 2eT(GL)PC. Moreover, 2eTPC specifically enhanced Tyr 579 and Tyr 857 phosphorylation in the PDGFR cytoplasmic domain, while the TMD mutant failed to support such phosphorylation. Finally, 2eTPC, but not 2eT(GL)PC, induced phosphorylation of Src and PI3 kinase (known downstream effectors of Tyr 579 phosphorylation) and promoted Src-mediated migration of NIH3T3 cells. Taken together, these data suggest that the TMD of a syndecan-2 specifically regulates receptor cytoplasmic domain function and subsequent downstream signaling events controlling cell behavior.     

Foldamers as Anticancer Therapeutics: Targeting Protein–Protein Interactions and the Cell Membrane

Targeting important protein–protein interactions involved in carcinogenesis or targeting the cell membrane of a cancer cell directly are just two of the ways in which foldamers (oligomeric molecules that fold into distinct shapes in solution) hold considerable potential in the treatment of cancer. From mimicking the local topography of the helical compound of interest by using covalently constrained foldamers to mimicking the topography of the natural helix such that the positions of key functional motifs are in an identical spatial orientation to match those presented by the original α‐helix, synthetic foldamers have been used to mimic the natural foldamers that interact with proteins or the cell membrane. These targeted approaches have become established over a timeframe of more than a decade, and they continue to be included in the assortment of cancer targets being studied and the arsenal of chemotherapy compounds in development. These approaches are reviewed herein.     

Conducting systems based on styrene–butadiene‐block–copolymers: Microstructural characterization

This work reports on the preparation and structural and electrical characterization of composites consisting of HSBR, polypropylene (PP), and carbon black (CB) blends, comparing the data obtained from more or less rich PP samples. Structural analysis provided evidence of the plasticizing or crosslinked effects on the properties of composites, when CB is present in the initial system, as well as of the excellent conducting properties of CB composites. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 84: 646–653, 2002; DOI 10.1002/app.10360     

Poly(ether sulfone) supported hybrid poly(vinyl alcohol)–maleic acid–silicone dioxide membranes for the pervaporation separation of ethanol–water mixtures

Microporous poly(ether sulfone) (PES) supported hybrid polymer–inorganic membranes were prepared by the crosslinking of poly(vinyl alcohol) (PVA), maleic acid (MA), and SiO₂ via an aqueous sol–gel route and a solution‐casting method. The membrane performance was tested for the pervaporation separation of ethanol–water mixtures from 20 to 60 °C with a feed ethanol concentration of 96 wt %. The membrane characterization results reveal that different SiO₂ loadings affected the crystallinity and roughness of the membranes. The PVA–MA–SiO₂ membrane containing 10 wt % SiO₂ showed that SiO₂ nanoparticles were well dispersed within the polymer matrix; this resulted in significant enhancements in both the flux and selectivity. The membrane achieved a high water permeability of 1202 g·μm·m^?2 h^?1 kPa^?1 and a selectivity of 1027 for the separation of a 96 wt % ethanol‐containing aqueous solution. This enhanced membrane performance might have been due to the dense crosslinking membrane network, increased free volume, and uniform distribution of SiO₂ nanoparticles. Both the water and ethanol fluxes increased with the feed water concentration and temperature. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134 , 44839.     

Preparation and properties of polysulfone–clay composite membranes

Porous membranes and dense films were prepared from polysulfone solutions in N‐methyl‐2‐pyrrolidone (NMP) containing different types and amounts of clay. Commercial clays supplied by Southern Clay, either unmodified (Cloisite Na) or organically modified (Cloisite 30B and Cloisite 93A), were used. The clay behavior in the organic solvent was dependent on the presence and type of the organic compatibilizer: Cloisite containing Na ions did not swell in NMP, whereas those with the organic compatibilizer swelled, though to a different degree. Electron microscopy observations were made to examine the clay dispersion in the membrane structure. At variance with Cloisite Na and Cloisite 93A formed microaggregates, Cloisite 30B yielded nanostructures composed of both single sheets and well‐ordered multilayer silicate clusters, which were characterized by an interlayer distance higher than that of the neat clay. The increase in the distance between the layers of Cloisite 30B was related to the formation of intercalated nanocomposites, whereas the presence of single sheets well distributed in the polymer matrix supported the occurrence of delaminated nanocomposites. The intercalation of the polymer into clay layers was confirmed with wide‐angle X‐ray diffractometry. The addition of Cloisite 30B to the casting solution influenced the phase‐separation process in the coagulation bath. Therefore, by the variation of the layered‐silicate concentration in the casting solution, membranes with different morphological structures and ultrafiltration properties were obtained. Cloisite 30B was also found to improve the wettability and mechanical properties of dense films. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 3637–3644, 2007     

Hybrid sol–gel membranes of polyacrylonitrile–tetraethoxysilane composites for gas permselectivity

Sol–gel reaction of tetraethoxysilane (TEOS) with fumed silica–polyacrylonitrile (PAN) membrane was carried out to prepare hybrid gas permeable membranes for oxygen and nitrogen separation. Various amounts of fumed silica microparticles with a few μm diameters were compounded in PAN–dimethylsulfoxide (DMSO) solution. After casting of the viscous compound solution on a flat sheet with 100 μm thickness, DMSO was evacuated under vacuum at 80°C. Then, the silica–PAN composite membranes were treated with TEOS for 1 day at 40°C in methanol. Air permeation was examined and compared in silica–PAN composite membranes with and without TEOS treatment. The latter hybrid membranes showed selective oxygen permeability, which depended on amounts of fumed silica in the membrane. The TEOS hybrid PAN membranes have a high ability of oxygen permselectivity for O₂/N₂ gas mixture with α(O₂/N₂) = 13–17, when the silica content was in the range of 13–20 wt %. This is attributed to siloxane network formation in hybrid silica–PAN composite membranes. Favorable siloxane network formation resulted in high oxygen permeability of the hybrid composite membranes. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 1752–1759, 2003     

Swelling behavior of PDMS–PMHS pervaporation membranes in ethyl acetate–water mixtures

Pervaporation composite membranes were prepared with a three‐layer structure: a PP support, a PEI microporous structure, and a PDMS–PMHS selective layer. Swelling tests were performed in water, ethyl acetate, and four different ethyl acetate–water mixtures, to calculate the diffusional and swelling parameters. Moreover, the dynamic–mechanical properties of the membranes were obtained before and after the swelling experiments, and their relaxation spectra were characterized with the Fuoss–Kirkwood equation and analyzed in terms of the free volume parameter. It was found that the ethyl acetate possesses high affinity with the composite membranes and that the absorption of these small molecules substantially modifies the viscoelastic behavior and structure of the active layer. The combination of swelling experiments and the study of the mechanical relaxations proved to be an appropriate technique to investigate the behavior of pervaporation membranes immersed in different mixtures. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 1384–1393, 2004     

Reversible Control of Exo‐ and Endo‐Budding Transitions in a Photosensitive Lipid Membrane

We have developed a method for the photomanipulation of lipid membrane morphology in which the shape of a vesicle can be switched by light through the use of a synthetic photosensitive amphiphile containing an azobenzene unit (KAON12). We prepared cell‐sized liposomes from KAON12 and 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) and conducted real‐time observations of vesicular transformation in the photosensitive liposome by phase‐contrast microscopy. Budding transitions—either budding toward the centre of the liposome (endo‐bud) or budding out of the liposome (exo‐bud)—could be controlled by light. We discuss the mechanism of this transformation in terms of the change in the effective membrane surface area due to photoisomerization of the constituent molecules.     

Influence of reactant concentration on formation of AgCl particles in PEO–PPO–PEO microemulsion and morphology and performance of AgCl–PMMA membranes

AgCl/poly(methyl methacrylate) (PMMA) organic–inorganic hybrid membrane has been synthesized by reverse microemulsion polymerization using triblock copolymer polyoxyethylene–polyoxypropylene–polyoxyethylene as surfactant and MMA as oil phase. The results by ultraviolet–visible spectrum, transmission electron microscopy, and scanning electron microscopy showed that small AgCl nanoparticles distributed well in the F127 microemulsions and hybrid membranes at low reactant concentration. AgCl nanoparticles in the microemulsion became smaller with increasing reactant concentration. However, AgCl nanoparticles aggregated obviously in hybrid membranes, when reactant concentration was more than 0.15 mol L^?1. The performance of different hybrid membranes for separation of the benzene and cyclohexane was measured. The results indicated that the separation performance of membrane was promoted obviously due to presence of more well‐dispersed AgCl particles in hybrid membranes. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Structural characterization of Bi–Zn–Nb–O cubic pyrochlores

Structure and dielectric properties of oxide dielectrics with the composition of Bi_1.5Zn_NNb_2.5−NO_8.5−1.5N (with N=0.73 to 1.20) have been studied. These samples were treated at 1050 °C for 4 h. The cubic pyrochlore phase was found to be predominant as from X-ray diffraction and Raman spectra analysis. Lattice constant of the cubic pyrochlore and dielectric constant of the sample have been found to increase with the increase of Zn content. A model of the structural defects has been proposed to explain the stabilization of the pyrochlore structure. The limitation of composition for the formation of single cubic pyrochlore phase has been attributed to the distribution of oxygen defects.     

Metal‐chelating properties of poly(2‐hydroxyethyl methacrylate–methacryloylamidohistidine) membranes

Metal‐chelating membranes have advantages as adsorbents in comparison with conventional beads because they are not compressible and they eliminate internal diffusion limitations. The aim of this study was to explore in detail the performance of poly(2‐hydroxyethyl methacrylate–methacryloylamidohistidine) [poly(HEMA–MAH)] membranes for the removal of three toxic heavy‐metal ions—Cd(II), Pb(II), and Hg(II)—from aquatic systems. The poly(HEMA–MAH) membranes were characterized with scanning electron microscopy and ¹H‐NMR spectroscopy. The adsorption capacity of the poly(HEMA–MAH) membranes for the selected heavy‐metal ions from aqueous media containing different amounts of these ions (30–500 mg/L) and at different pH values (3.0–7.0) was investigated. The adsorption capacity of the membranes increased with time during the first 60 min and then leveled off toward the equilibrium adsorption. The maximum amounts of the heavy‐metal ions adsorbed were 8.2, 31.5, and 23.2 mg/g for Cd(II), Pb(II), and Hg(II), respectively. The competitive adsorption of the metal ions was also studied. When the metal ions competed, the adsorbed amounts were 2.9 mg of Cd(II)/g, 14.8 mg of Pb(II)/g, and 9.4 mg of Hg(II)/g. The poly(HEMA–MAH) membranes could be regenerated via washing with a solution of nitric acid (0.01M). The desorption ratio was as high as 97%. These membranes were suitable for repeated use for more than three adsorption/desorption cycles with negligible loss in the adsorption capacity. The stability constants for the metal‐ion/2‐methacryloylamidohistidine complexes were calculated to be 3.47 × 10⁶, 7.75 × 10⁷, and 2.01 × 10⁷ L/mol for Cd(II), Pb(II), and Hg(II) ions, respectively, with the Ruzic method. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 1213–1219, 2005     

Preparation and pervaporation performances of fumed‐silica‐filled polydimethylsiloxane–polyamide (PA) composite membranes

Fumed‐silica‐filled polydimethylsiloxane (PDMS)–polyamide (PA) composite membranes were prepared by the introduction of hydrophobic fumed silica into a PDMS skin layer. The cross‐sectional morphology of these filled composite membranes was observed with scanning electron microscopy. Their pervaporation performances were tested with aqueous ethanol solutions at 30, 35, and 40°C. Increasing the amount of the fumed silica resulted in significantly enhanced ethanol permeability of the membranes. When the content of the fumed silica in the PDMS skin layer was 20 wt %, the ethanol permeability increased to nearly twice that of the unfilled PDMS–PA composite membrane. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Mechanoelectrical Conversion in Highly Ionic Conductive Solid‐State Polymer Electrolyte Membranes

A novel phenomenon of mechanoelectrical conversion in a flexible solid‐state polymer electrolyte membrane (PEM) is presented, hereafter denoted as flexoelectric effect. The flexoelectric coefficient (≈323 µC m^?1), that is, a measure of the converted mechanoelectrical energy, is the highest among all flexoelectric materials hitherto reported. It is proposed in this work that the flexoelectricity in PEMs operates based on electrical energy generation driven by ion polarization/depolarization across the PEM subjected to a pressure gradient during bending. Of particular interest is the phenomenon of polarity switching during bending, that is, reversal of the polarization direction with increasing succinonitrile (SCN) concentration (i.e., 10–20 wt%). The size disparity between the solvated cations and anions is attributed as the key factor in determining the polarization direction, which is responsible for the polarity switching. Of particular importance is that the present flexoelectric PEM itself is a key component of the solid‐state lithium ion battery and thus their integration opens up a new avenue for energy harvesting and storage devices.     

Optogenetic Control of PIP2 Interactions Shaping ENaC Activity

The activity of the epithelial Na⁺ Channel (ENaC) is strongly dependent on the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 binds two distinct cationic clusters within the N termini of β- and γ-ENaC subunits (βN1 and γN2). The affinities of these sites were previously determined using short synthetic peptides, yet their role in sensitizing ENaC to changes in PIP2 levels in the cellular system is not well established. We addressed this question by comparing the effects of PIP2 depletion and recovery on ENaC channel activity and intracellular Na⁺ levels [Na⁺]_i. We tested effects on ENaC activity with mutations to the PIP2 binding sites using the optogenetic system CIBN/CRY2-OCRL to selectively deplete PIP2. We monitored changes of [Na⁺]_i by measuring the fluorescent Na⁺ indicator, CoroNa Green AM, and changes in channel activity by performing patch clamp electrophysiology. Whole cell patch clamp measurements showed a complete lack of response to PIP2 depletion and recovery in ENaC with mutations to βN1 or γN2 or both sites, compared to wild type ENaC. Whereas mutant βN1 also had no change in CoroNa Green fluorescence in response to PIP2 depletion, γN2 did have reduced [Na⁺]_i, which was explained by having shorter CoroNa Green uptake and half-life. These results suggest that CoroNa Green measurements should be interpreted with caution. Importantly, the electrophysiology results show that the βN1 and γN2 sites on ENaC are each necessary to permit maximal ENaC activity in the presence of PIP2.     

Unraveling the Secrets of Protein–Metabolite Interactions

Saghatelian and colleagues recently introduced a global metabolite‐profiling approach that allows protein–metabolite interactions (PMI) to be identified. This approach represents an excellent strategy and valuable tool for unraveling the many secrets of the metabolome. The key features of the methodology will be summarized here.