Julien Tailhades

Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a commensal bacterium deficient in Crohn’s disease

Background Crohn’s disease (CD)-associated dysbiosis is characterised 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 dinitrobenzene sulfonic acid (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 non-polar residues. This last feature prevented the direct characterisation 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 nuclear factor (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. Conclusions A 15 kDa 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.

Proline-rich antimicrobial peptides: potential therapeutics against antibiotic-resistant bacteria.

The increasing resistance of pathogens to antibiotics causes a huge clinical burden that places great demands on academic researchers and the pharmaceutical industry for resolution. Antimicrobial peptides, part of native host defense, have emerged as novel potential antibiotic alternatives. Among the different classes of antimicrobial peptides, proline-rich antimicrobial peptides, predominantly sourced from insects, have been extensively investigated to study their specific modes of action. In this review, we focus on recent developments in these peptides. They show a variety of modes of actions, including mechanism shift at high concentration, non-lytic mechanisms, as well as possessing different intracellular targets and lipopolysaccharide binding activity. Furthermore, proline-rich antimicrobial peptides display the ability to not only modulate the immune system via cytokine activity or angiogenesis but also possess properties of penetrating cell membranes and crossing the blood brain barrier suggesting a role as potential novel carriers. Ongoing studies of these peptides will likely lead to the development of more potent antimicrobial peptides that may serve as important additions to the armoury of agents against bacterial infection and drug delivery.

The Efficacies of Cell-Penetrating Peptides in Accumulating in Large Unilamellar Vesicles Depend on their Ability To Form Inverted Micelles

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.

Synthesis of Peptide Alcohols on the Basis of an O–N Acyl‐Transfer Reaction

Peptides containing a C-terminal alcohol function (C-terminal peptide alcohols) constitute an important class of compounds. They exhibit a range of biological activities, such as the antibiotic properties of the peptaibols and the potent and unique biological activity of the metabolically stable somatostatin analogue octreotide (Sandostatin), which is used clinically for the diagnosis and treatment of a variety of neuroendocrine tumors and gastrointestinal disorders. Other examples of biologically active C-terminal peptide alcohols are the potent enkephalin analogue Tyr-d-Ala-GlyMePhe-Met(O)-ol, the gramicidins, and compounds isolated from Trichoderma species. Furthermore, peptide alcohols serve as precursors for the synthesis of peptide aldehydes, an important class of protease inhibitors and versatile synthetic intermediates. C-terminal peptide alcohols cannot be synthesized by conventional solid-phase peptide synthesis (SPPS) because of the absence of a free carboxylic group to attach to the resin. However, other SPPS methods have been developed that involve reductive cleavage of a peptide ester linkage, 9] the use of redox-sensitive resins, aminolysis of a peptide ester linkage with a bamino alcohol, or standard cleavage of a peptide alcohol ester linkage. Except when the chlorotrityl resin is used, anchoring of the hydroxy function requires derivatization of the linker or C-terminal bamino alcohol with a specific handle, such as that found in tetrahydropyranyl-based linkers, hemisuccinate linkers, 17] or polymeric diphenyldiazomethane; alternatively, activated resins can be used. There is still a strong need for new methodology for the preparation of peptide alcohols by the conventional 9fluorenylmethoxycarbonyl (Fmoc)/tBu SPPS strategy with commercially available resins that would enable the recovery of a nonprotected or totally protected peptide alcohol. According to these requirements, the trityl-type resin is the most promising. Unfortunately, the attachment of the alcohol group to this support remains difficult and proceeds in low yield. To overcome the drawbacks of these methods, we developed a new strategy based on an O–N acyl-transfer reaction (Scheme 1). A C-terminal b-amino alcohol residue was anchored to a trityl chloride resin through its b-amino

Cellular Disulfide Bond Formation in Bioactive Peptides and Proteins

Bioactive peptides play important roles in metabolic regulation and modulation and many are used as therapeutics. These peptides often possess disulfide bonds, which are important for their structure, function and stability. A systematic network of enzymes—a disulfide bond generating enzyme, a disulfide bond donor enzyme and a redox cofactor—that function inside the cell dictates the formation and maintenance of disulfide bonds. The main pathways that catalyze disulfide bond formation in peptides and proteins in prokaryotes and eukaryotes are remarkably similar and share several mechanistic features. This review summarizes the formation of disulfide bonds in peptides and proteins by cellular and recombinant machinery.

Intramolecular acyl transfer in peptide and protein ligation and synthesis

Intramolecular acyl transfer equilibrium in peptides and proteins has stimulated the development of new methodologies for ligation, aggregation suppression or difficult peptide synthesis. Native chemical ligation or aggregation suppression methodologies are based on an X‐to‐N acyl transfer of a peptide chain (X = S, O). The reverse reaction from N‐to‐X has led to exciting developments in solving key synthetic problems such as peptide thioester preparation using Fmoc/tBu strategy. Depending on the target peptide or protein, variations of these methods, which are also based on acyl transfer equilibriums, are now available.

Multimerization of a Proline-Rich Antimicrobial Peptide, Chex-Arg20, Alters Its Mechanism of Interaction with the Escherichia coli Membrane.

A3-APO, a de novo designed branched dimeric proline-rich antimicrobial peptide (PrAMP), is highly effective against a variety of in vivo bacterial infections. We undertook a selective examination of the mechanism for the Gram-negative Escherichia coli bacterial membrane interaction of the monomer (Chex-Arg20), dimer (A3-APO), and tetramer (A3-APO disulfide-linked dimer). All three synthetic peptides were effective at killing E. coli. However, the tetramer was 30-fold more membrane disruptive than the dimer while the monomer showed no membrane activity. Using flow cytometry and high-resolution fluorescent microscopy, it was observed that dimerization and tetramerization of the Chex-Arg20 monomer led to an alteration in the mechanism of action from non-lytic/membrane hyperpolarization to membrane disruption/depolarization. Our findings show that the membrane interaction and permeability of Chex-Arg20 was altered by multimerization.

Development of a single-chain peptide agonist of the relaxin-3 receptor using hydrocarbon stapling

Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist which will facilitate study of the function of relaxin-3.

How to unveil self-quenched fluorophores and subsequently map the subcellular distribution of exogenous peptides

Confocal laser scanning microscopy (CLSM) is the most popular technique for mapping the subcellular distribution of a fluorescent molecule and is widely used to investigate the penetration properties of exogenous macromolecules, such as cell-penetrating peptides (CPPs), within cells. Despite the membrane-association propensity of all these CPPs, the signal of the fluorescently labeled CPPs did not colocalize with the plasma membrane. We studied the origin of this fluorescence extinction and the overall consequence on the interpretation of intracellular localizations from CLSM pictures. We demonstrated that this discrepancy originated from fluorescence self-quenching. The fluorescence was unveiled by a “dilution” protocol, i.e. by varying the ratio fluorescent/non-fluorescent CPP. This strategy allowed us to rank with confidence the subcellular distribution of several CPPs, contributing to the elucidation of the penetration mechanism. More generally, this study proposes a broadly applicable and reliable method to study the subcellular distribution of any fluorescently labeled molecules.

Total Chemical Synthesis of an Intra-A-Chain Cystathionine Human Insulin Analogue with Enhanced Thermal Stability

Despite recent advances in the treatment of diabetes mellitus, storage of insulin formulations at 4 °C is still necessary to minimize chemical degradation. This is problematic in tropical regions where reliable refrigeration is not ubiquitous. Some degradation byproducts are caused by disulfide shuffling of cystine that leads to covalently bonded oligomers. Consequently we examined the utility of the non-reducible cystine isostere, cystathionine, within the A-chain. Reported herein is an efficient method for forming this mimic using simple monomeric building blocks. The intra-A-chain cystathionine insulin analogue was obtained in good overall yield, chemically characterized and demonstrated to possess native binding affinity for the insulin receptor isoform B. It was also shown to possess significantly enhanced thermal stability indicating potential application to next-generation insulin analogues.