Interaction of ‘toxic’ and ‘immunogenic’ A‐gliadin peptides with a membrane‐mimetic environment

Celiac disease (CD) is characterized by abnormally high concentrations of certain peptides in the small bowel. These peptides can be grouped in ‘toxic’ and ‘immunogenic’ classes, which elicit an innate immune response and an HLA‐mediated adaptive response, respectively. It is not clear on which molecular mechanisms responses to these different classes are based, but the 31–43 (P31–43) and the 56–68 (P56–68) A‐gliadin fragments are usually adopted as sequence representatives of toxic and immunogenic peptides, respectively. Here we report fluorescence experiments aiming to mimic the interaction of these peptides with the cell membrane surface by using sodium dodecyl sulphate (SDS) as a membrane‐mimetic medium. We show that P31–43 is able to bind SDS micelles in a way that resembles mixed micelle formation. On the other hand, no binding at all could be detected for P56–68. This different behaviour could be related to the paracellular or transcellular route through which gluten peptides may cross the intestinal epithelium, and open new insights into the pathogenetic mechanisms of CD. Copyright © 2009 John Wiley & Sons, Ltd.     

Regulation and Prediction of Enzyme Activity in Reversed Micelles

The rate of cholesterol oxidation has been studied in cholesterol oxidase containing reversed micellar media consisting of the surfactant cetyltrimethylammonium bromide (CTAB), the surfactant octanol, a buffered aqueous solution, and a variety of organic solvents. By varying the composition of the medium systematically it could be deduced that the rate of cholesterol oxidation obeys the same rules as described earlier for the conversion of apolar steroids by 20β-hydroxysteroid dehydrogenase in CTAB-hexanol-organic solvent reversed micelles (Hilhorst et al. 1984). The general applicability of these rules in optimizing biocatalysis in reversed micelles is discussed.     

Limited Proteolysis of Myelin Basic Protein in a System Mimetic of the Myelin Interlamellar Aqueous Space

Abstract: We have investigated the early steps of myelin basic protein (MBP) degradation in a membrane mimetic system (reverse micelles), resembling the interlamellar aqueous spaces where the protein is located in the myelin sheath. MBP, unfolded in buffer, refolds on incorporation into the micelles, resulting in reduced accessibility to three proteolytic enzymes, trypsin, cathespin D, and Staphylococcus aureus V8 protease, in comparison with aqueous solution. Eleven cleavage sites seen in buffer are removed from proteolytic attack in micellar solution. These sites delineate a protected protein domain displaying a potential β-sheet structure capable of interacting with the myelin membrane. An additional site not seen in buffer is attacked in the micelles. Experiments with a structure inducer, 15% 1-propanol in buffer, reveal that the refolding pattern of MBP in reverse micelles is specific to the membrane biomimetic system and is not produced by organic solvent per se. Micellar digestions of MBP generate long peptides, two of which, isolated after tryptic digestion, have been found to be immunodominant in multiple sclerosis patients. The findings suggest the structure induced in MBP by the micelles resembles that leading to production of the self-peptides recognized by T cells during proteolytic breakdown of MBP in autoimmune demyelinating diseases.     

Alkaline Titrations of poly(dG-dC)·poly(dG-dC): Microemulsion Versus Solution Behavior

Abstract

PolyGC was titrated with a strong base in the presence of increasing concentrations of NaCl (from 0.00 to 0.60M) either in water solution or with the polynucleotide solubilized in the aqueous core of reverse micelles, i.e., the cationic quaternary water-in-oil microemulsion CTAB/n-hexane/n-pentanol/water. The results for matched samples in the two media were compared. CD and UV spectroscopies and, for the solution experiments, pH measurements were used to follow the course of deprotonation. In both media the primary effect of the addition of base was denaturation of the polynucleotide, reversible by back-titration with a strong acid.

In solution, the apparent pK_a of the transition decreases with increasing the salt concentration and a roughly linear dependence of pK_a on p[NaCl] has been found. A parallel monotonic decay with ionic strength has been found in solution for R_OH, defined as the number of hydroxyl ions required per monomeric unit of polyGC to reach half-transition. By contrast, in microemulsion, R_OH has been found to be independent of the NaCl concentration (and 10 to 50 times lower than in solution). This result is proposed as an indirect evidence of the independence of pK_a on the salt concentration in microemulsion, where the pH cannot be measured. A sort of buffering effect of the positive charges on the micellar wall and of their counter-ions on the ionic strength could well explain this discrepancy of behavior in the two media.     

Single-spanning membrane protein insertion in membrane mimetic systems: role and localization of aromatic residues

Membrane protein insertion in the lipid bilayer is determining for their activity and is governed by various factors such as specific sequence motifs or key amino-acids. A detailed fluorescence study of such factors is exemplified with PMP1, a small (38 residues) single-membrane span protein that regulates the plasma membrane H⁺-ATPase in yeast and specifically interacts with phosphatidylserines. Such interactions may stabilize raft domains that have been shown to contain H⁺-ATPase. Previous NMR studies of various fragments have focused on the critical role of interfacial residues in the PMP1 structure and intermolecular interactions. The C-terminal domain contains a terminal Phe (F38), a single Trp (W28) and a single Tyr (Y25) that may act together to anchor the protein in the membrane. In order to describe the location and dynamics of W28 and the influence of Y25 on protein insertion within membrane, we carried out a detailed steady-state and time-resolved fluorescence study of the synthetic G13-F38 fragment and its Tyr-less mutant, Y25L in various membrane mimetic systems. Detergent micelles are conveniently used for this purpose. We used dodecylphosphocholine (DPC) in order to compare with and complement previous NMR results. In addition, dodecylmaltoside (DM) was used so that we could apply our recently described new quenching method by two brominated analogs of DM (de Foresta et al. 2002, Eur. Biophys. J. 31:185–97). In both systems, and in the presence and absence of Y25, W28 was shown to be located below but close to the polar headgroup region, as shown by its maximum emission wavelengths (λ_max), curves for the quenching of Trp by the brominated analogs of DM and bimolecular constants for quenching (k_q) by acrylamide. Results were interpreted by comparison with calibration data obtained with fluorescent model peptides. Time-resolved anisotropy measurements were consistent with PMP1 fragment immobilization within peptide-detergent complexes. We tentatively assigned the two major Trp lifetimes to the Trp (χ₁=60° and 180°) rotamers, based on the recent lifetime–rotamer correlation proposed for model cyclic peptides (Pan and Barkley 2004, Biophys J 86:3828–35). We also analyzed the role of the hydrophobic anchor, by comparing the micelle binding of fragments of various lengths including the synthesized full-length protein and detected peculiar differences for protein interaction with the polar headgroups of DM or DPC.     

Liquid-liquid extraction of lectin from Cratylia mollis seeds using reversed micelles

Extraction of lectin from seeds ofCratylia mollis, camaratu bean, with reversed micelles of 100 mM sodium di(2-ethylhexyl) sulfosuccinate/isooctane was performed firstly with affinity-purified lectin. The best conditions were extraction of the seed extract at pH 5 and back-extraction at pH 10, giving yields of 38% and 100%, respectively.     

Optimizing the Solution Conditions to Solve the Structure of the Connexin43 Carboxyl Terminus Attached to the 4th Transmembrane Domain in Detergent Micelles

Abstract

pH-mediated gating of Cx43 channels following an ischemic event is believed to contribute to the development of lethal cardiac arrhythmias. Studies using a soluble version of the Cx43 carboxyl-terminal domain (Cx43CT; S255–I382) have established the central role it plays in channel regulation; however, research in the authors' laboratory suggests that this construct may not be the ideal model system. Therefore, we have developed a more ‘native-like’ construct (Cx43CT attached to the 4th transmembrane domain [TM4-Cx43CT; G178–I382]) than the soluble Cx43CT to further investigate the mechanism(s) governing this regulation. Here, we utilize circular dichroism and nuclear magnetic resonance (NMR) were used to validate the TM4-Cx43CT for studying channel gating and optimize solution conditions for structural studies. The data indicate that, unlike the soluble Cx43CT, the TM4-Cx43CT is structurally responsive to changes in pH, suggesting the presence of the TM4 facilitates pH-induced structural alterations. Additionally, the optimal solution conditions for solving the NMR solution structure include 10% 2,2,2 trifluoroethanol and removal of the 2nd extracellular loop (G178-V196).     

Lipase-Catalyzed Synthesis of Fatty Acid Esters Useful in the Food Industry

Enzyme reactions are very attractive in food technology because they can be carried out under mild conditions and without toxic solvents and other catalysts. Lipases can esterify various alcohols with fatty acids. There are opportunities to synthesize useful compounds with special functions as food materials by using the catalytic function of lipase. Reverse micellar systems are discussed as reaction systems for lipases in organic solvents, especially in triacylglycerol synthesis using phosphatidylcholine as the surfactant. Syntheses of some amphiphilic substances including O-acyl-L-homoserine are also discussed.     

In vitro lipid peroxidation of intestinal bile salt-based nanoemulsions: Potential role of antioxidants

Abstract

Over the last decades, oxidative stress has been described as a deleterious phenomenon contributing to numerous noncommunicable diseases such as cardiovascular disease, diabetes, and cancers. As many authors ascribed the healthy effect of fruit and vegetable consumption mainly to their antioxidant contents, it has been hypothesized that their protection could occur from the gut. Therefore, the aim of this study was to develop an original and physiological model of nanoemulsions to study lipid peroxidation within the intestine and to assess the properties of potential antioxidants in this setting.

Several nanoemulsions were compared in terms of physical characteristics and reactivity to 2,2’-azobis-(2-amidinopropane) hydrochloride (AAPH)-induced oxidation. Formulations included different types of lipids, a detergent (a conjugated bile salt or sodium dodecyl sulfate) and, finally, lipophilic antioxidants. Hemin and myoglobin were also tested as relevant potential oxidants. Fatty acid (FA) peroxidation was monitored by gas chromatography while malondialdehyde and antioxidant contents were measured by HPLC.

Investigated nanoemulsions were composed of spherical or cylindrical mixed micelles, the latter being the least resistant to oxidation. In the experimental conditions, AAPH was the only efficient oxidant. Alpha-tocopherol and lutein significantly slowed FA degradation from 4 to 1 μM, respectively. On the contrary, beta-carotene did not show any protective capacity at 4 μM.

In conclusion, the tested nanoemulsions were appropriate to assess antioxidant capacity during the intestinal phase of digestion.     

Antioxidant properties of ganglioside micelles

Antioxidant activity of gangliosides GM1 and GT1b in the Fenton type of reaction was investigated by EPR spectroscopy using DMPO as a spin trap. Hydroxyl radical spin adduct signal intensity was significantly reduced in the presence of gangliosides at their micellar concentrations. Mean micellar hydrodynamic diameter was not changed, whereas significant changes in negative Zeta potential values were observed as evidenced by Zetasizer Nano ZS. This study showed that the primary mode of ganglioside action was not due to direct scavenging of OH·, but rather to the inhibition of hydroxyl radical formation. This phenomenon is related to the ability of ganglioside micelles to bind oppositely charged ferrous ions, thus reducing their concentration and consequently inhibiting OH· formation.