Physical and biophysical studies of membrane-membrane and membrane-protein interactions require well controlled model systems. We have developed a new method to obtain a single lipid bilayer floating in excess of water, in the vicinity of a fixed bilayer. It has been prepared in the gel phase, which is not biophysically relevant. The free bilayer has been precisely characterized by Neutron and X-Ray reflectivity. By increasing the temperature up to the melting transition of the lipid chains, one can observe a spectacular change in bilayer-bilayer distance. We attribute this swelling to the bilayer's interaction of Helfrich due to the bilayer fluctuations. Theoretical implications will be discussed, allowing us to measure the bending modulus of the membrane in a large range of temperatures. In order to characterize more precisely the fluctuations of the membrane we have developed X-Ray off-specular reflectivity leading to a direct measurement of the fluctuations spectrum of the membrane. As a conclusion we have obtained a free lipid bilayer, fluid and stable, biophysically relevant substrate for bilayer-bilayer or protein-bilayer interaction studies. This allows us to characterize the fluctuations spectrum of liquid membrane in a large range of temperatures. Enantioselective reactions starting from heterochiral mixtures of precursors might have been involved during the formation of homochiral biopolymers of life at prebiotic times. Since reactions in isotropic media would lead to heterochiral products, a way to obtain oligopeptides of homochiral sequences from racemic reactants would be through the assembly of the precursor molecules into ordered architectures followed by lattice-controlled reactions. Two-dimensional (2-D) self-assemblies of amphiphilic molecules formed on water surface provide an ideal medium. Racemates of reactive α-amino acid amphiphiles were designed to self-assemble, on water surface, into 2-D crystallites of three types: racemic compounds, enantiomorphous conglomerates and enantiomerically disordered solid-solutions. Such self-assemblies could undergo polymerization to yield mixtures of oligopeptides of enantiomeric composition controlled by the packing motif of the precursor crystalline phase. The 2-D self-assembly of various racemates and the reaction induced by an appropriate catalyst were studied, directly on water surface, by grazing incidence X-ray diffraction using synchrotron radiation. The enantiomeric composition of the oligopeptides collected from the interface was determined by matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry using deuterium enantio-labeled monomers. Or results show that polymerization within racemic 2-D crystallites can lead to the enhanced generation of oligopeptides with homochiral sequences through reaction between translation-related rather than glide-related molecules. When the reaction occurred primarily between glide-related molecules, heterochiral oligopeptides were obtained. The latter polymerization was taken advantage of t...

Pseudomonas entomophila is an entomopathogenic bacterium that is able to infect and kill Drosophila melanogaster upon ingestion. Its genome sequence suggests that it is a versatile soil bacterium closely related to Pseudomonas putida. The GacS/GacA two-component system plays a key role in P. entomophila pathogenicity, controlling many putative virulence factors and AprA, a secreted protease important to escape the fly immune response. P. entomophila secretes a strong diffusible hemolytic activity. Here, we showed that this activity is linked to the production of a new cyclic lipopeptide containing 14 amino acids and a 3-C 10 OH fatty acid that we called entolysin. Three nonribosomal peptide synthetases (EtlA, EtlB, EtlC) were identified as responsible for entolysin biosynthesis. Two additional components (EtlR, MacAB) are necessary for its production and secretion. The P. entomophila GacS/GacA two-component system regulates entolysin production, and we demonstrated that its functioning requires two small RNAs and two RsmA-like proteins. Finally, entolysin is required for swarming motility, as described for other lipopeptides, but it does not participate in the virulence of P. entomophila for Drosophila. While investigating the physiological role of entolysin, we also uncovered new phenotypes associated with P. entomophila, including strong biocontrol abilities.

Background Low concentrations of high-density lipoprotein cholesterol (HDL-C) represent a well-established cardiovascular risk factor. Paradoxically, extremely high HDL-C levels are equally associated with elevated cardiovascular risk, resulting in the U-shape relationship of HDL-C with cardiovascular disease. Mechanisms underlying this association are presently unknown. We hypothesised that the capacity of high-density lipoprotein (HDL) to acquire free cholesterol upon triglyceride-rich lipoprotein (TGRL) lipolysis by lipoprotein lipase underlies the non-linear relationship between HDL-C and cardiovascular risk. Methods To assess our hypothesis, we developed a novel assay to evaluate the capacity of HDL to acquire free cholesterol (as fluorescent TopFluor® cholesterol) from TGRL upon in vitro lipolysis by lipoprotein lipase. Results When the assay was applied to several populations markedly differing in plasma HDL-C levels, transfer of free cholesterol was significantly decreased in low HDL-C patients with acute myocardial infarction (−45%) and type 2 diabetes (–25%), and in subjects with extremely high HDL-C of >2.59 mmol/L (>100 mg/dL) (−20%) versus healthy normolipidaemic controls. When these data were combined and plotted against HDL-C concentrations, an inverse U-shape relationship was observed. Consistent with these findings, animal studies revealed that the capacity of HDL to acquire cholesterol upon lipolysis was reduced in low HDL-C apolipoprotein A-I knock-out mice and was negatively correlated with aortic accumulation of [3H]-cholesterol after oral gavage, attesting this functional characteristic as a negative metric of postprandial atherosclerosis. Conclusions Free cholesterol transfer to HDL upon TGRL lipolysis may underlie the U-shape relationship between HDL-C and cardiovascular disease, linking HDL-C to triglyceride metabolism and atherosclerosis.

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The amino acid p-benzoyl-L-phenylalanine, @-Bz)Phe, has been incorporated into substance P (SP), Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, to localize the agonist-binding domains of the human neurokinin-1 (NK-1) receptor overexpressed in a transfected mammalian cell line. The NK-1 -specific agonist [Pro9]SP was modified at position 8 by @-Bz)Phe and acylated at the N-terminus by a biotinyl sulfone reporter via a 5-aminopentanoyl spacer. After photolysis, the biotinyl sulfone moiety allowed easy and efficient removal of biotinyiated fragments from the complex incubation mixture with streptavidin-coated beads. Direct elution from the beads with the matrix used for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS), which was facilitated by saturation of streptavidin sites with biotin, and subsequent MALDI-TOF mass spectrometry analysis allowed identification of the NK-1 fragments obtained after photolysis and proteolytic digestion. Trypsin digestion and combined trypsinlstaphylococcus aureus V8 protease enzymatic cleavage established that the site of covalent attachment of the photolabelled SP resides in the second extracellular loop, Thr173 -Arg177. Cyanogen bromide cleavage shows that the probe is covalently attached to the methyl group of a methionine residue from human NK-1. These experiments identified Met174 as the modified residue.Keywords: tachykinin receptor; photolabelling ; substance P; Chinese hamster ovary cells ; mass spectrometry.Chimeric tachykinin NK-UNK-3 or NK-UNK-2 receptors and mutated neurokinin-1 (NK-1) receptors have been constructed to probe the binding domains of NK-1 agonists and antagonists [ 1 -151. Both extracellular domains (N-terminal residues and extracellular loops) and residues from the transmembrane segments [I, 3, 5, 7, 8, 12, 13, 151 have been identified as important determinants for the binding of substance P and specific NK-1 agonists. However, as is evident from the conflicting data, these results do not imply that these critical residues of the NK-1 receptor directly interact with the amino acids of substance P; these residues may only confer the proper folding to the binding site for the agonist in the protein [13, 151. Affinity labelling of the receptor using photoreactive ligands constitutes a complementary strategy to mutagenesis [16]. The use of photolabelled analogues of the ligand-incorporating reactive probes in various positions allows mapping of the agonistbinding site. Theoretically, this strategy seems more precise than mutagenesis for the determination of the amino acids that constitute the binding pocket in the protein. However, numerous photolabelling experiments should be performed to describe the foot-printing of the ligand bound to its receptor. It should be noted that (a) important residues of the ligand cannot usually be modified (loss of the binding potency); the residue that is easily replaced by a photoreactive probe may not contribute to the binding contacts with the protein; and (b) when a compromis...

16K prolactin (PRL) is the name given to the 16-kDa N-terminal fragment obtained by proteolysis of rat PRL by tissue extracts or cell lysates, in which cathepsin D was identified as the candidate protease. Based on its antiangiogenic activity, 16K PRL is potentially a physiological inhibitor of tumor growth. Full-length human PRL (hPRL) was reported to be resistant to cathepsin D, suggesting that antiangiogenic 16K PRL may be physiologically irrelevant in humans. In this study, we show that hPRL can be cleaved by cathepsin D or mammary cell extracts under the same conditions as described earlier for rat PRL, although with lower efficiency. In contrast to the rat hormone, hPRL proteolysis generates three 16K-like fragments, which were identified by N-terminal sequencing and mass spectrometry as corresponding to amino acids 1-132 (15 kDa), 1-147 (16.5 kDa), and 1-150 (17 kDa). Biochemical and mutagenetic studies showed that the species-specific digestion pattern is due to subtle differences in primary and tertiary structures of rat and human hormones. The antiangiogenic activity of N-terminal hPRL fragments was assessed by the inhibition of growth factor-induced thymidine uptake and MAPK activation in bovine umbilical endothelial cells. Finally, an N-terminal hPRL fragment comigrating with the proteolytic 17-kDa fragment was identified in human pituitary adenomas, suggesting that the physiological relevance of antiangiogenic N-terminal hPRL fragments needs to be reevaluated in humans.

The ins and outs of peptides: The internalization efficiencies of three cell‐penetrating peptides (CPPs; penetratin, (Arg)9, and Tat48–59) have been measured by MALDI‐TOF MS (see scheme). The method leads to direct detection of the CPPs and allows unambiguous discrimination between membrane‐bound and internalized CPP. Furthermore, the cellular uptake efficiencies of several CPPs can be compared in a single experiment.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry of polystyrenes prepared by 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO)-mediated living free radical polymerization has been performed using two different matrices. A complete assignment of the observed peaks could be proposed, owing to the experimental resolution which allowed us to display the isotopic distribution. With the 1,8-dihydroxy-9(10H)-anthracenone (dithranol)/silver trifluoroacetate system, a major part of the charged chains undergoes gas phase fragmentation during the analysis. This phenomenon, only minor for conventionnally prepared polystyrene, is particularly enhanced when the chains contain a TEMPO-based alkoxyamine end group. In contrast, the dead chains with no alkoxyamine end group are properly detected. When using the 2,5-dihydroxybenzoic acid (DHB) matrix without added salt, only protonation occurs involving the alkoxyamine functionality. Only the TEMPO-capped polystyrene chains are observed and no fragmentation occurs; the dead chains which have no protonation site are not detected. This still allows determination of the nature of the polymer headgroup and gives an insight into the initiation mechanism. Thermally self-initiated polystyrene in the presence of TEMPO contains predominantly a 4-phenyl-1,2,3,4-tetrahydro-1-naphthyl headgroup. When dibenzoyl peroxide (BPO) is used as an initiator at 130 °C in the presence of TEMPO ([TEMPO]/[BPO] = 1.2), both benzoyloxy and 4-phenyl-1,2,3,4-tetrahydro-1-naphthyl headgroups are detected. When an alkoxyamine initiator is used, only the fragment derived from this initiator is observed and the extent of thermal initiation seems to be reduced by comparison with the use of dibenzoyl peroxide initiator. These results can be explained by the enhanced formation of 4-phenyl-1,2,3,4-tetrahydro-1-naphthyl radical in the presence of free TEMPO.