Characterization of SLC26A9 in patients with CF-like lung disease.

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.

Modeling of non-covalent complexes of the cell-penetrating peptide CADY and its siRNA cargo.

CADY is a cell-penetrating peptide spontaneously making non-covalent complexes with Short interfering RNAs (siRNAs) in water. Neither the structure of CADY nor that of the complexes is resolved. We have calculated and analyzed 3D models of CADY and of the non-covalent CADY-siRNA complexes in order to understand their formation and stabilization. Data from the ab initio calculations and molecular dynamics support that, in agreement with the experimental data, CADY is a polymorphic peptide partly helical. Taking into consideration the polymorphism of CADY, we calculated and compared several complexes with peptide/siRNA ratios of up to 40. Four complexes were run by using molecular dynamics. The initial binding of CADYs is essentially due to the electrostatic interactions of the arginines with siRNA phosphates. Due to a repetitive arginine motif (XLWR(K)) in CADY and to the numerous phosphate moieties in the siRNA, CADYs can adopt multiple positions at the siRNA surface leading to numerous possibilities of complexes. Nevertheless, several complex properties are common: an average of 14±1 CADYs is required to saturate a siRNA as compared to the 12±2 CADYs experimentally described. The 40 CADYs/siRNA that is the optimal ratio for vector stability always corresponds to two layers of CADYs per siRNA. When siRNA is covered by the first layer of CADYs, the peptides still bind despite the electrostatic repulsion. The peptide cage is stabilized by hydrophobic CADY-CADY contacts thanks to CADY polymorphism. The analysis demonstrates that the hydrophobicity, the presence of several positive charges and the disorder of CADY are mandatory to make stable the CADY-siRNA complexes.

Insight into the cellular uptake mechanism of a secondary amphipathic cell-penetrating peptide for siRNA delivery.

Delivery of siRNA 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 siRNA 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 siRNA and binds phospholipids tightly, mainly through electrostatic interactions. Binding to siRNA 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.

Enzymatic interesterification of anhydrous milk fat with rapeseed and/or linseed oil: oxidative stability.

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.

Mutational analysis of the TRE2 oncogene encoding an inactive RabGAP.

The TRE2 oncoprotein is structurally related to the RabGAP (GTPase-activating protein) family. However, TRE2 seems enzymatically inactive. Two regions are important for its lack of GAP activity. First, the TBC domain, forming the catalytically active domain of RabGAPs, is non-functional in the oncoprotein. Also involved in TRE2 inactivity is the 93-aa region flanking the TBC domain on the C-terminal side. In order to identify the residues responsible for non-functionality, we performed hydrophobic cluster analysis of the oncoprotein sequence, combined with secondary structure prediction, receptor-binding domain analysis, and a tilted peptide calculation. These analyses were complemented with site-directed and random mutagenesis experiments. On the basis of our data, we hypothesize that the lack of secondary structure of the region flanking the TBC domain in TRE2 may explain why this region plays a role in the lack of GAP activity, even when a potentially functional TBC domain is present.

Mode of membrane interaction and fusogenic properties of a de novo transmembrane model peptide depend on the length of the hydrophobic core.

Model peptides composed of alanine and leucine residues are often used to mimic single helical transmembrane domains. Many studies have been carried out to determine how they interact with membranes. However, few studies have investigated their lipid-destabilizing effect. We designed three peptides designated KALRs containing a hydrophobic stretch of 14, 18, or 22 alanines/leucines surrounded by charged amino acids. Molecular modeling simulations in an implicit membrane model as well as attenuated total reflection-Fourier transform infrared analyses show that KALR is a good model of a transmembrane helix. However, tryptophan fluorescence and attenuated total reflection-Fourier transform infrared spectroscopy indicate that the extent of binding and insertion into lipids increases with the length of the peptide hydrophobic core. Although binding can be directly correlated to peptide hydrophobicity, we show that insertion of peptides into a membrane is determined by the length of the peptide hydrophobic core. Functional studies were performed by measuring the ability of peptides to induce lipid mixing and leakage of liposomes. The data reveal that whereas KALR14 does not destabilize liposomal membranes, KALR18 and KALR22 induce 40 and 50% of lipid-mixing, and 65 and 80% of leakage, respectively. These results indicate that a transmembrane model peptide can induce liposome fusion in vitro if it is long enough. The reasons for the link between length and fusogenicity are discussed in relation to studies of transmembrane domains of viral fusion proteins. We propose that fusogenicity depends not only on peptide insertion but also on the ability of peptides to destabilize the two leaflets of the liposome membrane.

Tilted peptides: the history.

Nature has selected peptide motifs for protein functions. It is clear that specific sequence motifs can identify families of enzymes. These sequence motifs are one dimensional signatures and nature has also developed two dimension motifs which cannot be read in the one dimension of sequence language but can be detected in the three dimensional properties of a secondary structure. One of such motifs is tilted peptides. They do not correspond to any consensus of sequence but correspond to a consensus motif where hydrophobicity balance is used as a functional device. In the nineteen eighties, the first tilted peptide was deciphered from the sequence of a virus fusion protein by molecular modelling. It was described as a protein fragment hydrophobic enough to insert into a membrane but too short to span it. The fragment exhibited an asymmetric distribution of hydrophobicity along the helix long axis and hence, was unable to lie parallel or perpendicular to a membrane surface but adopted an orientation in between. Hydrophobicity motif was a very new and very challenging concept and tilted peptides were rapidly found to be involved in several mechanisms of virus fusion. They were also found to be involved in protein secretion and future studies could establish their involvement in the destabilization of 3D protein structures and in the alpha to beta transconformations, which drive the generation of amyloid deposits.

Prediction of peptide structure: how far are we?

Rational design of peptides is a challenge, which would benefit from a better knowledge of the rules of sequence-structure-function relationships. Peptide structures can be approached by spectroscopy and NMR techniques but data from these approaches too frequently diverge. Structures can also be calculated in silico from primary sequence information using three algorithms: Pepstr, Robetta, and PepLook. The most recent algorithm, PepLook introduces indexes for evaluating structural polymorphism and stability. For peptides with converging experimental data, calculated structures from PepLook and, to a lesser extent from Pepstr, are close to NMR models. The PepLook index for polymorphism is low and the index for stability points out possible binding sites. For peptides with divergent experimental data, calculated and NMR structures can be similar or, can be different. These differences are apparently due to polymorphism and to different conditions of structure assays and calculations. The PepLook index for polymorphism maps the fragments encoding disorder. This should provide new means for the rational design of peptides.

Role of the lid hydrophobicity pattern in pancreatic lipase activity.

Pancreatic lipase is a soluble globular protein that must undergo structural modifications before it can hydrolyze oil droplets coated with bile salts. The binding of colipase and movement of the lipase lid open access to the active site. Mechanisms triggering lid mobility are unclear. The *KNILSQIVDIDGI* fragment of the lid of the human pancreatic lipase is predicted by molecular modeling to be a tilted peptide. Tilted peptides are hydrophobicity motifs involved in membrane fusion and more globally in perturbations of hydrophobic/hydrophilic interfaces. Analysis of this lid fragment predicts no clear consensus of secondary structure that suggests that its structure is not strongly sequence determined and could vary with environment. Point mutations were designed to modify the hydrophobicity profile of the [240-252] fragment and their consequences on the lipase-mediated catalysis were tested. Two mutants, in which the tilted peptide motif was lost, also have poor activity on bile salt-coated oil droplets and cannot be reactivated by colipase. Conversely, one mutant in which a different tilted peptide is created retains colipase dependence. These results suggest that the tilted hydrophobicity pattern of the [240-252] fragment is neither important for colipase binding to lipase, nor for interfacial binding but is important to trigger the maximal catalytic efficiency of lipase in the presence of bile salt.