Steroid structural requirements for interaction of ostreolysin, a lipid-raft binding cytolysin, with lipid monolayers and bilayers

AbstractOstreolysin, a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), recognizes and binds specifically to membrane domains enriched in cholesterol and sphingomyelin (or saturated phosphatidylcholine). These events, leading to permeabilization of the membrane, suggest that a cholesterol-rich liquid-ordered membrane phase, which is characteristic of lipid rafts, could be its possible binding site. In this work, we present effects of ostreolysin on membranes containing various steroids. Binding and membrane permeabilizing activity of ostreolysin was studied using lipid mono- and bilayers composed of sphingomyelin combined, in a 1/1 molar ratio, with natural and synthetic steroids (cholesterol, ergosterol, β-sitosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, cholesteryl acetate, and 5-cholesten-3-one). Binding to membranes and lytic activity of the protein are both shown to be dependent on the intact sterol 3β-OH group, and are decreased by introducing additional double bonds and methylation of the steroid skeleton or C17-isooctyl chain. The activity of ostreolysin mainly correlates with the ability of the steroids to promote formation of liquid-ordered membrane domains, and is the highest with cholesterol-containing membranes. Furthermore, increasing the cholesterol concentration enhanced ostreolysin binding in a highly cooperative manner, suggesting that the membrane lateral distribution and accessibility of the sterols are crucial for the activity of this new member of cholesterol-dependent cytolysins

A comparative study of the actions of alkylpyridinium salts from a marine sponge and related synthetic compounds in rat cultured hippocampal neurones

Background: Polymeric alkylpyridinium salts (poly-APS), are chemical defences produced by marine sponges including Reniera sarai. Poly-APS have previously been shown to effectively deliver macromolecules into cells. The efficiency of this closely follows the ability of poly-APS to form transient pores in membranes, providing strong support for a pore-based delivery mechanism. Recently, water soluble compounds have been synthesised that are structurally related to the natural polymers but bear a different number of pyridinium units. These compounds may share a number of bio-activities with poly-APS. Using electrophysiology, calcium imaging and 1,6-diphenyl-1,3,5-hexatriene imaging, the pore forming properties of poly-APS and four related synthetic oligomers have been tested on primary cultured rat hippocampal neurones.Results: Acute application of poly-APS (0.5 μg/ml), reduced membrane potential, input resistance and suppressed action potential firing. Poly-APS evoked inward cation currents with linear current-voltage relationships similar to actions of pore formers on other cell types. Poly-APS (0.005-5 μg/ml) also produced Ca2+ transients in ∼41% of neurones. The dose-dependence of poly-APS actions were complex, such that at 0.05 μg/ml and 5 μg/ml poly-APS produced varying magnitudes of membrane permeability depending on the order of application. Data from surface plasmon resonance analysis suggested accumulation of poly-APS in membranes and subsequent enhanced poly-APS binding. Even at 10-100 fold higher concentrations, none of the synthetic compounds produced changes in electrophysiological characteristics of the same magnitude as poly-APS. Of the synthetic oligomers tested compounds 1 (monomeric) and tetrameric 4 (5-50 μg/ ml) induced small transient currents and 3 (trimeric) and 4 (tetrameric) produced significant Ca2+ transients in hippocampal neurones.Conclusion: Poly-APS induced pore formation in hippocampal neurones andsuch pores were transient, with neurones recovering from exposure to these polymers. Synthetic structurally related oligomers were not potent pore formers when compared to poly-APS and affected a smaller percentage of the hippocampal neurone population. Poly-APS may have potential as agents for macromolecular delivery into CNS neurones however; the smaller synthetic oligomers tested in this study show little potential for such use. This comparative analysis indicated that the level of polymerisation giving rise to the supermolecular structure in the natural compounds, is likely to be responsible for the activity here reported.</p

A comparative study of the actions of alkylpyridinium salts from a marine sponge and related synthetic compounds in rat cultured hippocampal neurones

BACKGROUND: Polymeric alkylpyridinium salts (poly-APS), are chemical defences produced by marine sponges including Reniera sarai. Poly-APS have previously been shown to effectively deliver macromolecules into cells. The efficiency of this closely follows the ability of poly-APS to form transient pores in membranes, providing strong support for a pore-based delivery mechanism. Recently, water soluble compounds have been synthesised that are structurally related to the natural polymers but bear a different number of pyridinium units. These compounds may share a number of bio-activities with poly-APS. Using electrophysiology, calcium imaging and 1,6-diphenyl-1,3,5-hexatriene imaging, the pore forming properties of poly-APS and four related synthetic oligomers have been tested on primary cultured rat hippocampal neurones. RESULTS: Acute application of poly-APS (0.5 μg/ml), reduced membrane potential, input resistance and suppressed action potential firing. Poly-APS evoked inward cation currents with linear current-voltage relationships similar to actions of pore formers on other cell types. Poly-APS (0.005–5 μg/ml) also produced Ca(2+ )transients in ~41% of neurones. The dose-dependence of poly-APS actions were complex, such that at 0.05 μg/ml and 5 μg/ml poly-APS produced varying magnitudes of membrane permeability depending on the order of application. Data from surface plasmon resonance analysis suggested accumulation of poly-APS in membranes and subsequent enhanced poly-APS binding. Even at 10–100 fold higher concentrations, none of the synthetic compounds produced changes in electrophysiological characteristics of the same magnitude as poly-APS. Of the synthetic oligomers tested compounds 1 (monomeric) and tetrameric 4 (5–50 μg/ml) induced small transient currents and 3 (trimeric) and 4 (tetrameric) produced significant Ca(2+ )transients in hippocampal neurones. CONCLUSION: Poly-APS induced pore formation in hippocampal neurones and such pores were transient, with neurones recovering from exposure to these polymers. Synthetic structurally related oligomers were not potent pore formers when compared to poly-APS and affected a smaller percentage of the hippocampal neurone population. Poly-APS may have potential as agents for macromolecular delivery into CNS neurones however; the smaller synthetic oligomers tested in this study show little potential for such use. This comparative analysis indicated that the level of polymerisation giving rise to the supermolecular structure in the natural compounds, is likely to be responsible for the activity here reported

Allostery and Conformational Dynamics in cAMP-binding Acyltransferases

Mycobacteria harbor unique proteins that regulate protein lysine acylation in a cAMP-regulated manner. These lysine acyltransferases from Mycobacterium smegmatis (KATms) and Mycobacterium tuberculosis (KATmt) show distinctive biochemical properties in terms of cAMP binding affinity to the N-terminal cyclic nucleotide binding domain and allosteric activation of the C-terminal acyltransferase domain. Here we provide evidence for structural features in KATms that account for high affinity cAMP binding and elevated acyltransferase activity in the absence of cAMP. Structure-guided mutational analysis converted KATms from a cAMP-regulated to a cAMP-dependent acyltransferase and identified a unique asparagine residue in the acyltransferase domain of KATms that assists in the enzymatic reaction in the absence of a highly conserved glutamate residue seen in Gcn5-related N-acetyltransferase-like acyltransferases. Thus, we have identified mechanisms by which properties of similar proteins have diverged in two species of mycobacteria by modifications in amino acid sequence, which can dramatically alter the abundance of conformational states adopted by a protein

Utility of Chromatographic and Spectroscopic Techniques for a Detailed Characterization of Poly(styrene‑<i>b</i>‑isoprene) Miktoarm Star Copolymers with Complex Architecture

We analyzed various miktoarm star copolymers of the PS­(PI)_<i>x</i> type (<i>x</i> = 2, 3, 5, 7), which consist of one long polystyrene (PS) arm (82 or 105 kDa) and various numbers of short polyisoprene (PI) arms (from 11.3 to 39.7 kDa), prepared by anionic polymerization and selective chlorosilane chemistry. The length of the PI arm in stars decreases with the number of arms, so that the chemical compositions of all PS­(PI)_<i>x</i> samples were comparable. Our aim was to determine the purity of samples and to identify exactly the constituents of individual samples. For this purpose we used a variety of separation techniques (size-exclusion chromatography (SEC), reversed-phase liquid-adsorption chromatography (RP-LAC), and two-dimensional liquid chromatography (2D-LC)) and characterization techniques (UV-MALS-RI multidetection SEC system, NMR, and MALDI-TOF MS). The best separation and identification of the samples’ constituents were achieved by RP-LAC, which separates macromolecules according to their chemical composition, and a subsequent analysis of the off-line collected fractions from the RP-C18 column by SEC/UV-MALS-RI multidetection system. The results showed that all PS­(PI)_<i>x</i> samples contained the homo-PS and homo-PI in minor amounts and the high-molar-mass (PS)_<i>y</i>(PI)_<i>z</i> (<i>y</i> > 1) species, the content of which is higher in the samples PS­(PI)₅ and PS­(PI)₇ than in the samples PS­(PI)₂ and PS­(PI)₃. The major constituent of the PS­(PI)₂ sample was the one with the predicted structure. On the other hand, the major components of the PS­(PI)_<i>x</i> (<i>x</i> = 3, 5, and 7) samples were the stars consisting of a smaller number of PI arms than predicted from the functionalities of chlorosilane coupling agents. These results are in agreement with the average chemical composition of samples determined by proton NMR spectroscopy and characterization of the constituents by MALDI-TOF MS