Background Crohn's disease (CD) associated dysbiosis is characterized by a loss of Faecalibacterium prausnitzii, whose culture supernatant exerts an anti-inflammatory effect both in vitro and in vivo. However, the chemical nature of the anti-inflammatory compounds has not yet been determined. Methods Peptidomic analysis using mass spectrometry was applied to F. prausnitzii supernatant. Anti-inflammatory effects of identified peptides were tested in vitro directly on intestinal epithelial cell lines and on cell lines transfected with a plasmid construction coding for the candidate protein encompassing these peptides. In vivo, the cDNA of the candidate protein was delivered to the gut by recombinant Lactic Acid Bacteria to prevent DNBS-colitis in mice. Results The seven peptides, identified in the F. prausnitzii culture supernatants, derived from a single Microbial Anti-inflammatory Molecule (MAM), a protein of 15 kDa and comprising 53% of nonpolar residues. This last feature prevented the direct characterization of the putative anti-inflammatory activity of MAM-derived peptides. Transfection of MAM cDNA in epithelial cells led to a significant decrease in the activation of the NF-κB pathway with a dose-dependent effect. Finally, the use of a food-grade bacterium, Lactococcus lactis, delivering a plasmid encoding MAM was able to alleviate DNBS-induced colitis in mice. Conclusion A 15kDa protein with anti-inflammatory properties is produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein is able to inhibit the NF-κB pathway in intestinal epithelial cells and to prevent colitis in an animal model.
The ability of diamond nanoparticles (nanodiamonds, NDs) to deliver small interfering RNA (siRNA) into Ewing sarcoma cells is investigated with a view to the possibility of in-vivo anticancer nucleic-acid drug delivery. siRNA is adsorbed onto NDs that are coated with cationic polymer. Cell uptake of NDs is demonstrated by taking advantage of the NDs' intrinsic fluorescence from embedded color-center defects. Cell toxicity of these coated NDs is shown to be low. Consistent with the internalization efficacy, a specific inhibition of EWS/Fli-1 gene expression is shown at the mRNA and protein level by the ND-vectorized siRNA in a serum-containing medium.
The three-dimensional structure of substance P has been studied by 'H-NMR, (500 MHz), and by circular dichroism (CD) in different solvents. The analysis of the different NMR parameters suggest that substance P adopts a rather extended structure in dimethylsulfoxide and pyridine. In water, besides the aggregation phenomenon, the monomeric substance P presents a complex conformational equilibrium. The addition of sodium dodecylsulfate to the aqueous solution induces, as shown by CD spectroscopy, a preferential a-helical conformation. And in methanol three structural conclusions may be drawn: the flexibility of the N-terminal Arg-Pro-Lys, the a-helical structure of Pro4-Gln5-Gln6-Phe7-Phe' and the interaction of the C-terminal carboxamide with the primary amides from both glutamines.The amino acid sequences of the most commonly studied tachykinins are listed in Table 1. These peptides share a common C-terminal pentapeptide Phe-Xaa-Gly-Leu-Met-NH2 as well as a similar spectrum of biological activities. Their peripheral actions include hypotension, vasodilatation, salivation and contraction of various smooth muscles. The difference in the rank order of potency of these tachykinins on various pharmacological bioassays [l, 21, behavioral [3 -51 and electrophysiological [6, 71 studies has suggested the presence of different subtypes of tachykinin receptors. Binding data, obtained with labelled analogues of these peptides on various preparations from the peripheral [8] and the central nervous systems [9 -171, have indicated the existence of at least two distinct high-affinity binding sites. In the central system we have shown [18] that substance P (SP) has the highest potency for one of these binding sites, while NKB seems to be the endogenous ligand for the other [18-201. Since the third mammalian tachykinin NKA [19, 211 has a very reduced binding potency for these two binding sites it is likely that a third one, which is more specific for NKA, exists. These results are in agreement with the recent biochemical [22] and pharmacological [22] (D. Regoli, personal communication) data obtained by other groups.From the structure/activity and structure/affinity relationship studies it has been established that the minimum sequence which manifests activity is contained in the C-terminal part of the molecule and that the various potencies originate from the heterogeneity of the N-terminal sequences of the tachykinins. Thus, these common C-terminal amino acids might be regarded as the 'message sequence', the different N-terminal sequences, being the 'address sequences', are therefore related to the specificity of each peptide for its own receptor. What- ever the process of recognition, for example a lock and key or a zipper mechanism, two structural hypotheses have to be considered. On the one hand, in spite of the homology of the primary structure of the C-terminal part, the various Nterminal sequences confer to each peptide a completely different three-dimensional structure ; on the other hand, the spatial structures of the C...
Cell-penetrating peptides (CPPs) can cross the cell membrane and are widely used to deliver bioactive cargoes inside cells. The cargo and the CPP are often conjugated through a disulfide bridge with the common acceptation that this linker is stable in the extracellular biological medium and should not perturb the internalization process. However, with the use of thiol-specific reagents combined with mass spectrometry (as a quantitative method to measure intracellular concentrations of peptides) and confocal microscopy (as a qualitative method to visualize internalized peptides) analyses, we could show that, depending on the peptide sequence, thiol/disulfide exchange reactions could happen at the cell surface. These exchange reactions lead to the reduction of disulfide conjugates. In addition, it was observed that not only disulfide- but also thiol-containing peptides could cross-react with cell-surface thiols. The peptides cross-linked by thiol-containing membrane proteins were either trapped in the membrane or further internalized. Therefore, a new route of cellular uptake was unveiled that is not restricted to CPPs: a protein kinase C peptide inhibitor that is not cell permeant could cross cell membranes when an activated cysteine (with a 3-nitro-2-pyridinesulfenyl moiety) was introduced in its sequence.
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...
The overlapping biological behaviors between some cell penetrating peptides (CPPs) and antimicrobial peptides (AMPs) suggest both common and different membrane interaction mechanisms. We thus explore the capacity of selected CPPs and AMPs to reorganize the planar distribution of binary lipid mixtures by means of differential scanning calorimetry (DSC). Additionally, membrane integrity assays and circular dichroism (CD) experiments were performed. Two CPPs (Penetratin and RL16) and AMPs belonging to the dermaseptin superfamily (Drs B2 and C-terminal truncated analog [1-23]-Drs B2 and two plasticins DRP-PBN2 and DRP-PD36KF) were selected. Herein we probed the impact of headgroup charges and acyl chain composition (length and unsaturation) on the peptide/lipid interaction by using binary lipid mixtures. All peptides were shown to be alpha-helical in all the lipid mixtures investigated, except for the two CPPs and [1-23]-Drs B2 in the presence of zwitterionic lipid mixtures where they were rather unstructured. Depending on the lipid composition and peptide sequence, simple binding to the lipid surface that occur without affecting the lipid distribution is observed in particular in the case of AMPs. Recruitments and segregation of lipids were observed, essentially for CPPs, without a clear relationship between peptide conformation and their effect in the lipid lateral organization. Nonetheless, in most cases after initial electrostatic recognition between the peptide charged amino acids and the lipid headgroups, the lipids with the lowest phase transition temperature were selectively recruited by cationic peptides while those with the highest phase transition were segregated. Membrane activities of CPPs and AMPs could be thus related to their preferential interactions with membrane defects that correspond to areas with marked fluidity. Moreover, due to the distinct membrane composition of prokaryotes and eukaryotes, lateral heterogeneity may be differently affected by cationic peptides leading to either uptake or/and antimicrobial activities.
In this study, the direct translocation of cell-penetrating peptides (CPPs) into large unilamellar vesicles (LUVs) was shown to be rapid for all the most commonly used CPPs. This translocation led within a few minutes to intravesicular accumulation up to 0.5 mM, with no need for a transbilayer potential. The accumulation of CPPs inside LUVs was found to depend on CPP sequence, CPP extravesicular concentration and phospholipid (PL) composition, either in binary or ternary mixtures of PLs. More interestingly, the role of anionic phospholipid flip-flopping in the translocation process was ascertained. CPPs enhanced the flipping of PLs, and the intravesicular CPP accumulation directly correlated with the amount of anionic PLs that had been transferred from the external to the internal leaflet of the LUV bilayer, thus demonstrating the transport of peptide/lipid complexes as inverted micelles.
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