INTRODUCTION:Phosphatidylinositol-4,5-bisphosphate (PIP2) is a cofactor necessary for the activity of KCNQ1 channels. Some Long QT mutations of KCNQ1, including R243H, R539W and R555C have been shown to decrease KCNQ1 interaction with PIP2. A previous study suggested that R539W is paradoxically less sensitive to intracellular magnesium inhibition than the WT channel, despite a decreased interaction with PIP2. In the present study, we confirm this peculiar behavior of R539W and suggest a molecular mechanism underlying it.METHODS AND RESULTS:COS-7 cells were transfected with WT or mutated KCNE1-KCNQ1 channel, and patch-clamp recordings were performed in giant-patch, permeabilized-patch or ruptured-patch configuration. Similar to other channels with a decreased PIP2 affinity, we observed that the R243H and R555C mutations lead to an accelerated current rundown when membrane PIP2 levels are decreasing. As opposed to R243H and R555C mutants, R539W is not more but rather less sensitive to PIP2 decrease than the WT channel. A molecular model of a fragment of the KCNQ1 C-terminus and the membrane bilayer suggested that a potential novel interaction of R539W with cholesterol stabilizes the channel opening and hence prevents rundown upon PIP2 depletion. We then carried out the same rundown experiments under cholesterol depletion and observed an accelerated R539W rundown that is consistent with this model.CONCLUSIONS:We show for the first time that a mutation may shift the channel interaction with PIP2 to a preference for cholesterol. This de novo interaction wanes the sensitivity to PIP2 variations, showing that a mutated channel with a decreased affinity to PIP2 could paradoxically present a slowed current rundown compared to the WT channel. This suggests that caution is required when using measurements of current rundown as an indicator to compare WT and mutant channel PIP2 sensitivity.

Introduction Phosphatidylinositol-4,5-bisphosphate (PIP2) is a cofactor necessary for the activity of KCNQ1 channels. Some Long QT mutations of KCNQ1, including R243H, R539W and R555C have been shown to decrease KCNQ1 interaction with PIP2. A previous study suggested that R539W is paradoxically less sensitive to intracellular magnesium inhibition than the WT channel, despite a decreased interaction with PIP2. In the present study, we confirm this peculiar behavior of R539W and suggest a molecular mechanism underlying it. Methods and Results COS-7 cells were transfected with WT or mutated KCNE1-KCNQ1 channel, and patch-clamp recordings were performed in giant-patch, permeabilized-patch or ruptured-patch configuration. Similar to other channels with a decreased PIP2 affinity, we observed that the R243H and R555C mutations lead to an accelerated current rundown when membrane PIP2 levels are decreasing. As opposed to R243H and R555C mutants, R539W is not more but rather less sensitive to PIP2 decrease than the WT channel. A molecular model of a fragment of the KCNQ1 C-terminus and the membrane bilayer suggested that a potential novel interaction of R539W with cholesterol stabilizes the channel opening and hence prevents rundown upon PIP2 depletion. We then carried out the same rundown experiments under cholesterol depletion and observed an accelerated R539W rundown that is consistent with this model. Conclusions We show for the first time that a mutation may shift the channel interaction with PIP2 to a preference for cholesterol. This de novo interaction wanes the sensitivity to PIP2 variations, showing that a mutated channel with a decreased affinity to PIP2 could paradoxically present a slowed current rundown compared to the WT channel. This suggests that caution is required when using measurements of current rundown as an indicator to compare WT and mutant channel PIP2 sensitivity.

Diffuse bronchiectasis is a common problem in respiratory clinics. We hypothesized that mutations in the solute carrier 26A9 (SLC26A9) gene, encoding for a chloride (Cl(-)) transporter mainly expressed in lungs, may lead to defects in mucociliary clearance. We describe two missense variants in the SLC26A9 gene in heterozygote patients presenting with diffuse idiopathic bronchiectasis : p.Arg575Trp, identified in a patient also heterozygote for p.Phe508del in the CFTR gene; and p.Val486Ile. Expression of both mutants in Xenopus laevis oocytes abolished SLC26A9-mediated Cl(-) conductance without decreasing protein membrane expression. Coexpression of CFTR with SLC26A9-p.Val486Ile resulted in a significant increase in the Cl(-) current induced by PKA stimulation, similar to that obtained in oocytes expressing CFTR and SLC26A9-WT. In contrast, coexpression of CFTR with SLC26A9-p.Arg575Trp inhibited SLC26A9-enhanced CFTR activation upon PKA. Further structure-function analyses led us to propose a site encompassing Arg575 in the SLC26A9-STAS domain for CFTR-SLC26A9 interaction. We hypothesize that SLC26A9-p.Arg575Trp prevented SLC26A9-mediated functional activation of CFTR by altering SLC26A9-CFTR interaction. Although we cannot confirm that these mutations by themselves are deleterious, we propose that they trigger the pathogenic role of a single CFTR mutation and provide insight into a novel mechanism of Cl(-) transport alteration across the respiratory mucosa, based on functional inhibition of CFTR.

The secondary amphipathic peptide CADY forms stable positively charged nanoparticles with short interfering RNA (siRNA), which involves both electrostatic and hydrophobic interactions. CADY/siRNA self-assembling nanoparticles adopt a "raspberry"-like architecture cemented together by a matrix of free peptides. These nanoparticles are nontoxic and promote efficient delivery of siRNA in challenging primary cell lines.

We extended the use of Peplook, an in silico procedure for the prediction of three‐dimensional (3D) models of linear peptides to the prediction of 3D models of cyclic peptides and thanks to theab initiocalculation procedure, to the calculation of peptides with non‐proteinogenic amino acids. Indeed, such peptides cannot be predicted by homology or threading. We compare the calculated models with NMR and X‐ray models and for the cyclic peptides, with models predicted by other in silico procedures (Pep‐Fold and I‐Tasser). For cyclic peptides, on a set of 38 peptides, average root mean square deviation of backbone atoms (BB‐RMSD) was 3.8 and 4.1 Å for Peplook and Pep‐Fold, respectively. The best results are obtained with I‐Tasser (2.5 Å) although evaluations were biased by the fact that the resolved Protein Data Bank models could be used as template by the server. Peplook and Pep‐Fold give similar results, better for short (up to 20 residues) than for longer peptides. For peptides with non‐proteinogenic residues, performances of Peplook are sound with an average BB‐RMSD of 3.6 Å for ‘non‐natural peptides’ and 3.4 Å for peptides combining non‐proteinogenic residues and cyclic structure. These results open interesting possibilities for the design of peptidic drugs. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

Point mutation of nearby residues in ion channels can be associated with diametrically opposed clinical phenotypes despite the mutant channels exhibit similar biophysical characteristics. Hence a characterization of the channel structure/function at the amino-acid scale is required for better understanding of channel genotype/phenotype relationship. R555C and R539W KCNQ1 mutant channels are a good illustration of this idea: R555C mutation is associated with a fruste form of type 1 long QT syndrome, whereas R539W mutation is associated with sudden death.

Delivery of si RNA remains a major limitation to their clinical application, and several technologies have been proposed to improve their cellular uptake We recently described a peptide-based nanoparticle system for efficient delivery of si RNA into primary cell lines. CADY. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA and improves their cellular uptake independently of the endosomal pathway. In the present work, we have combined molecular modeling, spectroscopy, and membrane interaction approaches in order to gain further insight into CADY/siRNA particle mechanism of interaction with biological membrane. We demonstrate that CADY forms stable complexes with si RNA and binds phospholipids tightly, mainly through electrostatic interactions. Binding to si RNA or phospholipids triggers a conformational transition of CADY from an unfolded state to an alpha-helical structure, thereby stabilizing CADY/siRNA complexes and improving their interactions with cell membranes Therefore, we propose that CADY cellular membrane interaction is driven by its structural polymorphism which enables stabilization of both electrostatic and hydrophobic contacts with surface membrane proteoglycan and phospholipids.

Caveolin-1 has a segment of hydrophobic amino acids comprising approximately residues 103-122. We have performed an in silico analysis of the conformational preference of this segment of caveolin-1 using PepLook. We find that there is one main group of stable conformations corresponding to a hydrophobic U bent model that would not traverse the membrane. Furthermore, the calculations predict that substituting the Pro(110) residue with an Ala will change the conformation to a straight hydrophobic helix that would traverse the membrane. We have expressed the P110A mutant of caveolin-1, with a FLAG tag at the N terminus, in HEK 293 cells. We evaluate the topology of the proteins with confocal immunofluorescence microscopy in these cells. We find that FLAG tag at the N terminus of the wild type caveolin-1 is not reactive with antibodies unless the cell membrane is permeabilized with detergent. This indicates that in these cells, the hydrophobic segment of this protein is not transmembrane but takes up a bent conformation, making the protein monotopic. In contrast, the FLAG tag at the N terminus of the P110A mutant is equally exposed to antibodies, before and after membrane permeabilization. We also find that the P110A mutation causes a large reduction of endocytosis of caveolae, cellular lipid accumulation, and lipid droplet formulation. In addition, we find that this mutation markedly reduces the ability of caveolin-1 to form structures with the characteristic morphology of caveolae or to partition into the detergent-resistant membranes of these cells. Thus, the single Pro residue in the membrane-inserting segment of caveolin-1 plays an important role in both the membrane topology and localization of the protein as well as its functions.

Transfers of cargoes into cells by means of carrier peptides are multi-steps biological phenomenon the mechanisms of which are unclear. We here discuss bases of realistic in silico molecular modeling approaches of the formation of non-covalent complexes considering CPPs and cargo diversities.

We compare mean force potential values of a large series of PDB models of proteins and peptides and find that, either as monomers or polymers, proteins longer than 200–250 residues have equivalent MFP values that are averaged to –65±3kcal/aa. This value is named the standard or stability value. The standard value is reached irrespective of sequences and 3D folds. Peptides are too short to follow the rule and frequently exist as populations of conformers; one exception is peptides in amyloid fibrils. Fibrils surpass the standard value in accordance with their uppermost stability. In parallel, we calculate median MFP values of amino acids in stably folded PDB models of proteins: median values vary from –25 for Gly to –115kcal/aa for Trp. These median values are used to score primary sequences of proteins: all sequences converge to a mean value of −63.5±2.5kcal/aa, i.e., only 1.5kcal less than the folded model standard. Sequences from unfolded proteins have lower values. This supports the conclusion that sequences carry in an important message and more specifically that diversity of amino acids in sequences is mandatory for stability. We also use the median amino acid MFP to score residue stability in 3D folds. This demonstrates that 3D folds are compromises between fragments of high and fragments of low scores and that functional residues are often but not always in the extreme score values. The approach opens to possibilities of evaluating any 3D model and of detecting functional residues and should help in conducting mutation assays.

Biological membranes are complex structures composed largely of proteins and lipids. These components have very different structural and physical properties and consequently they do not form a single homogeneous mixture. Rather components of the mixture are more enriched in some regions than in others. This can be demonstrated with simple lipid mixtures that spontaneously segregate components so as to form different lipid phases that are immiscible with one another. The segregation of molecular components of biological membranes also involves proteins. One driving force that would promote the segregation of membrane components is the preferential interaction between a protein and certain lipid components. Among the varied lipid components of mammalian membranes, the structure and physical properties of cholesterol is quite different from that of other major membrane lipids. It would therefore be expected that in many cases proteins would have very different energies of interaction with cholesterol vs. those of other membrane lipids. This would be sufficient to cause segregation of components in membranes. The factors that facilitate the interaction of proteins with cholesterol are varied and are not yet completely understood. However, there are certain groups that are present in some proteins that facilitate interaction of the protein with cholesterol. These groups include saturated acyl chains of lipidated proteins, as well as certain amino acid sequences. Although there is some understanding as to why these particular groups favour interaction with cholesterol, our knowledge of these molecular features is not sufficiently developed to allow for the design of agents that will modify such binding.

Blends of anhydrous milk fat (AMF) and linseed oil (70:30) and of AMF, rapeseed oil (RO), and linseed oil (LO) (70:20:10) were submitted to enzymatic interesterification. The oxidative stabilities of the blends, the interesterified (IE) blends, and IE blends with 50 ppm of alpha-tocopherol added as antioxidant were studied. Samples were stored in open flasks at 60, 25, and 4 degrees C and periodically submitted to peroxide, p-anisidine, and TBA value determinations and UV measurement at 232 and 268 nm. The analysis of volatile compounds was carried out by SPME for the samples stored at 60 degrees C. Peroxides appeared to be the only significant oxidation products after 12 weeks of storage at 4 degrees C. As expected, the binary blends (BB) were more sensitive to oxidation than the ternary blends (TB). The BB were associated with increased volatile emission compared to the TB. Interesterification led to variable effects on the oxidation of fat mixtures, depending on composition and temperature (beneficial effect on BB, at both 25 and 60 degrees C, and a rather neutral effect on TB). The IE blends exhibited higher volatile release prior to aging. A pro-oxidant effect of alpha-tocopherol addition was observed at 25 degrees C on both BB and TB. At 60 degrees C, an antioxidant effect was observed on TB.

Introduction Chemical and/or physical modification of oils and fats are commonly used by food industry to widen their range of applications (1,2). Lipase-catalysed interesterification of anhydrous milk fat (AMF) and various vegetable oils is now a well documented procedure (3-7). The purpose of this technique is to produce original structured fats with properties different from a simple blending, that may be used as spreads or introduced into pastry. The new fats contain higher amounts of polyunsaturated fatty acids (PUFA) than butter, which provides health benefits (8,9). To our knowledge only a few authors associated AMF fractionation with blending and interesterification (10,11), although this combination may be used to increase the ratio of vegetable oil in blends and thus the PUFA content of the product. The compositional changes occurring during the lipase-catalysed interesterification of AMF/rapeseed oil (RO) and AMF stearin fraction (AMFSF)/RO blends were described in the first part of this study. In the present and second part are reported the resulting changes in physical properties, especially the melting behaviour through solid fat content (SFC), dropping point (DP) and fusion profiles by differential scanning calorimetry (DSC).