H-2Kd-restricted antigenic peptides share a simple binding motif.

We have defined structural features that are apparently important for the binding of four different, unrelated antigenic epitopes to the same major histocompatibility complex (MHC) class I molecule, H-2Kd. The four epitopes are recognized in the form of synthetic peptides by cytotoxic T lymphocytes of the appropriate specificity. By analysis of the relative potency of truncated peptides, we demonstrated that for each of the four epitopes, optimal antigenic activity was present in a peptide of 9 or 10 amino acid residues. A comparison of the relative competitor activity of the different-length peptides in a functional competition assay, as well as in a direct binding assay based on photoaffinity labeling of the Kd molecule, indicated that the enhanced potency of the peptides upon reduction in length was most likely due to a higher affinity of the shorter peptides for the Kd molecule. A remarkably simple motif that appears to be important for the specific binding of Kd-restricted peptides was identified by the analysis of peptides containing amino acid substitutions or deletions. The motif consists of two elements, a Tyr in the second position relative to the NH2 terminus and a hydrophobic residue with a large aliphatic side chain (Leu, Ile, or Val) at the COOH-terminal end of the optimal 9- or 10-mer peptides. We demonstrated that a simple peptide analogue (AYP6L) that incorporates the motif can effectively and specifically interact with the Kd molecule. Moreover, all of the additional Kd-restricted epitopes defined thus far in the literature contain the motif, and it may thus be useful for the prediction of new epitopes recognized by T cells in the context of this MHC class I molecule.

The final N-terminal trimming of a subaminoterminal proline-containing HLA class I-restricted antigenic peptide in the cytosol is mediated by two peptidases.

The proteasome produces MHC class I-restricted antigenic peptides carrying N-terminal extensions, which are trimmed by other peptidases in the cytosol or within the endoplasmic reticulum. In this study, we show that the N-terminal editing of an antigenic peptide with a predicted low TAP affinity can occur in the cytosol. Using proteomics, we identified two cytosolic peptidases, tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, that trimmed the N-terminal extensions of the precursors produced by the proteasome, and led to a transient enrichment of the final antigenic peptide. These peptidases acted either sequentially or redundantly, depending on the extension remaining at the N terminus of the peptides released from the proteasome. Inhibition of these peptidases abolished the CTL-mediated recognition of Ag-expressing cells. Although we observed some proteolytic activity in fractions enriched in endoplasmic reticulum, it could not compensate for the loss of tripeptidyl peptidase II/puromycin-sensitive aminopeptidase activities.

Multivalent display of the antimicrobial peptides BP100 and BP143

Carbohydrates are considered as promising templates for the display of multiple copies of antimicrobial peptides. Herein, wedescribe the design and synthesis of chimeric structures containing two or four copies of the antimicrobial peptidesKKLFKKILKYL-NH2 (BP100) and KKLfKKILKYL-NH2 (BP143) attached to the carbohydrate template cyclodithioerythritol(cDTE) or α-D-galactopyranoside (Galp). The synthesis involved the preparation of the corresponding peptide aldehyde followedby coupling to an aminooxy-functionalized carbohydrate template. After purification, the multivalent display systems were obtainedin high purities (90–98%) and in good yields (42–64%). These compounds were tested against plant and human pathogenic bacteriaand screened for their cytotoxicity on eukaryotic cells. They showed lower MIC values than the parent peptides against the bacteriaanalyzed. In particular, the carbopeptides derived from cDTE and Galp, which contained two or four copies of BP100, respectively,were 2- to 8-fold more active than the monomeric peptide against the phytopathogenic bacteria. These results suggest thatpreassembling antimicrobial peptides to multimeric structures is not always associated with a significant improvement of theactivity. In contrast, the carbopeptides synthesized were active against human red blood cells pointing out that peptide preassemblyis critical for the hemolytic activity. Notably, peptide preassembly resulted in an enhanced bactericidal effect

Soluble MHC-peptide complexes containing long rigid linkers abolish CTL-mediated cytotoxicity.

Soluble MHC-peptide (pMHC) complexes induce intracellular calcium mobilization, diverse phosphorylation events, and death of CD8+ CTL, given that they are at least dimeric and co-engage CD8. By testing dimeric, tetrameric, and octameric pMHC complexes containing spacers of different lengths, we show that their ability to activate CTL decreases as the distance between their subunit MHC complexes increases. Remarkably, pMHC complexes containing long rigid polyproline spacers (> or =80 A) inhibit target cell killing by cloned S14 CTL in a dose- and valence-dependent manner. Long octameric pMHC complexes abolished target cell lysis, even very strong lysis, at nanomolar concentrations. By contrast, an altered peptide ligand antagonist was only weakly inhibitory and only at high concentrations. Long D(b)-gp33 complexes strongly and specifically inhibited the D(b)-restricted lymphocytic choriomeningitis virus CTL response in vitro and in vivo. We show that complications related to transfer of peptide from soluble to cell-associated MHC molecules can be circumvented by using covalent pMHC complexes. Long pMHC complexes efficiently inhibited CTL target cell conjugate formation by interfering with TCR-mediated activation of LFA-1. Such reagents provide a new and powerful means to inhibit Ag-specific CTL responses and hence should be useful to blunt autoimmune disorders such as diabetes type I.

Antibody-mediated and cellular immune responses induced in naive volunteers by vaccination with long synthetic peptides derived from the Plasmodium vivax circumsporozoite protein.

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate. We describe the characterization of specific immune responses induced in 21 malaria-naive volunteers vaccinated with long synthetic peptides derived from the CS protein formulated in Montanide ISA 720. Both antibody- and cell-mediated immune responses were analyzed. Antibodies were predominantly of IgG1 and IgG3 isotypes, recognized parasite proteins on the immunofluorescent antibody test, and partially blocked sporozoite invasion of hepatoma cell lines in vitro. Peripheral blood mononuclear cells from most volunteers (94%) showed IFN-γ production in vitro upon stimulation with both long signal peptide and short peptides containing CD8+ T-cell epitopes. The relatively limited sample size did not allow conclusions about HLA associations with the immune responses observed. In summary, the inherent safety and tolerability together with strong antibody responses, invasion blocking activity, and the IFN-γ production induced by these vaccine candidates warrants further testing in a phase II clinical trial.

Exploring the space of histidine containing dipeptides in search of novel efficient RCS sequestering agents.

The study reports a set of forty proteinogenic histidine-containing dipeptides as potential carbonyl quenchers. The peptides were chosen to cover as exhaustively as possible the accessible chemical space, and their quenching activities toward 4-hydroxy-2-nonenal (HNE) and pyridoxal were evaluated by HPLC analyses. The peptides were capped at the C-terminus as methyl esters or amides to favor their resistance to proteolysis and diastereoisomeric pairs were considered to reveal the influence of configuration on quenching. On average, the examined dipeptides are less active than the parent compound carnosine (βAla + His) thus emphasizing the unfavorable effect of the shortening of the βAla residue as confirmed by the control dipeptide Gly-His. Nevertheless, some peptides show promising activities toward HNE combined with a remarkable selectivity. The results emphasize the beneficial role of aromatic and positively charged residues, while negatively charged and H-bonding side chains show a detrimental effect on quenching. As a trend, ester derivatives are slightly more active than amides while heterochiral peptides are more active than their homochiral diastereoisomer. Overall, the results reveal that quenching activity strongly depends on conformational effects and vicinal residues (as evidenced by the reported QSAR analysis), offering insightful clues for the design of improved carbonyl quenchers and to rationalize the specific reactivity of histidine residues within proteins.

Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp

We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37oC, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme.

Méthodologie pour la synthèse combinatoire d’azapeptides: application à la synthèse d’analogues aza-GHRP-6 en tant que ligands du récepteur CD36

Les azapeptides sont des mimes peptidiques où le carbone alpha d’un ou de plusieurs acides aminés est remplacé par un atome d’azote. Cette modification tend à stabiliser une conformation en repliement beta en raison de la répulsion électronique entre les paires d’électrons libres des atomes d’azote adjacents et de la géométrie plane de l’urée. De plus, le résidu semicarbazide a une meilleure résistance face aux protéases en plus d’être chimiquement plus stable qu’une liaison amide. Bien que les propriétés des azapeptides en fassent des mimes peptidiques intéressants, leurs méthodes de synthèses font appel à la synthèse laborieuse d’hydrazines substituées en solution. Le peptide sécréteur d’hormone de croissance 6 (GHRP-6, His-D-Trp-Ala-Trp-D-Phe-Lys-NH2) est un hexapeptide synthétique qui possède une affinité pour deux récepteurs distincts: les récepteurs GHS-R1a et CD36. Les travaux effectués au cours de mon doctorat qui seront détaillés dans cet ouvrage visent à atteindre deux objectifs: (1) le développement d’analogues du peptide GHRP-6 sélectif à un seul récepteur et (2) la mise au point d’une nouvelle méthodologie pour la synthèse combinatoire d’azapeptides. En réponse au premier objectif, la synthèse parallèle de 49 analogues aza-GHRP-6 a été effectuée et certains candidats sélectifs au récepteur CD36 ont été identifiés. L’étude de leurs propriétés anti-angiogéniques, effectuée par nos collaborateurs, a également permis d’identifier des candidats intéressants pour le traitement potentiel de la dégénérescence maculaire liée à l’âge. Une nouvelle approche pour la synthèse combinatoire d’azapeptides, faisant appel à l’alkylation et la déprotection chimiosélective d’une sous-unité semicarbazone ancrée sur support solide, a ensuite été développée. La portée de cette méthodologie a été augmentée par la découverte de conditions permettant l’arylation régiosélective de cette sous-unité semicarbazone, donnant accès à treize nouveaux dérivés aza-GHRP-6 possédant des résidus aza-arylglycines aux positions D-Trp2 et Trp4. L’élaboration de conditions propices à l’alkylation et la déprotection chimiosélective de la semicarbazone a donné accès à une variété de chaînes latérales sur l’acide aminé « aza » préalablement inaccessibles. Nous avons, entre autres, démontré qu’une chaîne latérale propargyl pouvait être incorporée sur l’acide aminé « aza ». Tenant compte de la réactivité des alcynes, nous avons ensuite élaboré des conditions réactionnelles permettant la formation in situ d’azotures aromatiques, suivie d’une réaction de cycloaddition 1,3-dipolaire sur support solide, dans le but d’obtenir des mimes de tryptophane. Sept analogues du GHRP-6 ont été synthétisés et testés pour affinité au récepteur CD36 par nos collaborateurs. De plus, nous avons effectué une réaction de couplage en solution entre un dipeptide possédant un résidu aza-propargylglycine, du paraformaldehyde et une variété d’amines secondaires (couplage A3) afin d’accéder à des mimes rigides d’aza-lysine. Ces sous-unités ont ensuite été incorporées sur support solide afin de générer sept nouveaux azapeptides avec des dérivés aza-lysine à la position Trp4 du GHRP-6. Enfin, une réaction de cyclisation 5-exo-dig a été développée pour la synthèse de N-amino imidazolin-2-ones en tant que nouveaux mimes peptidiques. Leur fonctionnalisation par une série de groupements benzyliques à la position 4 de l’hétérocycle a été rendue possible grâce à un couplage Sonogashira précédant la réaction de cyclisation. Les propriétés conformationnelles de cette nouvelle famille de composés ont été étudiées par cristallographie aux rayons X et spectroscopie RMN d’un tétrapeptide modèle. L’activité biologique de deux mimes peptidiques, possédant un résidu N-amino-4-méthyl- et 4-benzyl-imidazolin-2-one à la position Trp4 du GHRP-6, a aussi été examinée. L’ensemble de ces travaux devrait contribuer à l’avancement des connaissances au niveau des facteurs structurels et conformationnels requis pour le développement d’azapeptides en tant que ligands du récepteur CD36. De plus, les résultats obtenus devraient encourager davantage l’utilisation d’azapeptides comme peptidomimétiques grâce à leur nouvelle facilité de synthèse et la diversité grandissante au niveau de la chaîne latérale des acides aminés « aza ».

Synthesis of antimicrobial peptides derived from BP100 and BPC194

In the present PhD thesis we studied the solid-phase peptide synthesis of antimicrobial peptides derived from the lead peptides BP100 and BPC194. First, peptides derived from BP100 containing D-amino acids at different positions of the sequences were prepared. Moreover, peptidotriazoles derived from BP100 were also synthesized containing the triazole ring at the side-chain of different amino acids. Then, we proceeded to perform studies for the synthesis of multivalent peptides derived from BPC194. To achieve this objective, the synthesis of cyclic peptides containig a triazole ring at amino acids side-chain with different elongations was carried out. Finally, we prepared various carbopeptides containing 2 and 4 units of BP100 and/or its derivatives. The evaluation of the biological activity allowed the identification of active sequences against the economically important phytopathogenic bacteria and fungi and not toxic against eukaryotic cells.

Savory peptides present in Moromi obtained from soy sauce fermentation of yellow soybean

The presence of savory peptides in moromi has been investigated. Moromi was prepared by fermenting yellow soybean using Aspergillus oryzae as the starter at the first step (mold fermentation) and 20% brine solution at the next step (brine fermentation). The moromi was then ultrafiltered stepwise using membranes with MW cut-offs of 10,000, 3,000, and 500 Da, respectively. The fraction with MW < 500 Da was chromatographed using Sephadex G-25 SF to yield four fractions, 1-4. Analysis of soluble peptides, NaCl content, alpha-amino nitrogen, amino acid composition, peptide profile using CE coupled with DAD, taste profile and free glutamic acid content, were performed for each fraction. Fraction 2 contained a relatively high total glutamic acid content, but a relatively low free glutamic acid content and had the highest umami taste. This fraction also had more peptides containing non-aromatic amino acids than the other fractions. The peptides present in fraction 2 may play a role, at least in part, in its intense umami taste.

Supramolecular parallel beta-sheet and amyloid-like fibril forming peptides using delta-aminovaleric acid residue

Four terminally blocked tripeptides containing delta-aminovaleric acid residue self-assemble to form supramolecular beta-sheet structures as are revealed from their FT-IR data. Single crystal X-ray diffraction studies of two representative peptides also show that they form parallel beta-sheet structures. Self-aggregation of these beta-sheet forming peptides leads to the formation of fibrillar structures, as is evident from scanning electron microscopic (SEM) and transmission electron microscopic (TEM) images. These peptide fibrils bind to a physiological dye, Congo red and exhibit a typical green-gold birefringence under polarized light, showing close resemblance to neurodegenerative disease causing amyloid fibrils. (c) 2005 Elsevier Ltd. All rights reserved.

Conformational and self-assembly studies of helix forming hexapeptides containing two alpha-amino isobutyric acids

Single crystal X-ray diffraction studies reveal that three hexapeptides with general formula Boc-Ile-Aib-Xx-Ile-Aib-Yy-OMe, where Xx and Yy are Leu in peptide I, Len and Phe in peptide II, and Phe and Leu in peptide III, respectively, adopt equivalent conformations that can be described as mixed 3(10)/alpha-helice with two 4 -> 1 and two 5 -> 1 intramolecular N-H center dot center dot center dot O=C H-bonds. The peptides do not generate any helixterminating Schellman motif despite having Aib at the penultimate position from C-terminus. In the crystalline state, the helices are packed in head-to-tail fashion through intermolecular hydrogen bonds to create supramolecular helical structures. The CD Studies of the three hexapeptides in acetonitrile indicate that they are folded in well-developed 3(10)-helical structures. NMR studies of peptide I in CDCl3 also suggest the formation of a homogeneous 3 m-helical structure. The field emission scanning electron microscopic (FE-SEM) images of peptide 11 in the solid state reveal a non-twisted ribbon-like morphology, which is formed through lateral association of non-twisted filaments. (c) 2007 Elsevier Ltd. All rights reserved.

Conformational heterogeneity of tripeptides containing Boc-Leu-Aib as corner residues in the solid state

A critical analysis of single crystal X-ray diffraction studies on a series of terminally protected tripeptides containing a centrally positioned Aib (alpha-aminoisobutyric acid) residue has been reported. For the tripeptide series containing Boc-Ala-Aib as corner residues, all the reported peptides formed distorted type II beta-turn structures. Moreover, a series of Phe substituted analogues ( tripeptides with Boc-Phe-Aib) have also shown different beta-turn conformations. However, the Leu-modified analogues (tripeptides with Boc-Leu-Aib) disrupt the concept of beta-turn formation and adopt various conformations in the solid state. X-ray crystallography sheds some light on the conformational heterogeneity at atomic resolution. (c) 2007 Elsevier Ltd. All rights reserved.

Design of supramolecular beta-sheet forming beta-turns containing aromatic rings and non-coded alpha-aminoisobutyric acid and the formation of flat fibrillar structures through self-assembly

Single crystal X-ray diffraction studies show that the three designed tripeptides Boc-Leu-Aib-m-NA-NO2 (I), Boc-Phe-Aib-m-NA-NO2 (II) and Boc-Pro-Aib-m-ABA-OMe (III) (Aib, -aminoisobutyric acid; m-NA, m-nitroaniline; m-ABA, m-aminobenzoic acid; Boc, t-butyloxycarbonyl) containing aromatic rings in the backbones adopt -turn structures that are self-assembled through intermolecular hydrogen bonds and van der Waals interactions to create layers of -sheets. Solvent-dependent NMR titration and CD studies show that the -turn structures of the peptides also exist in the solution phase. The field emission scanning electron microscopic and transmission electron microscopic images of the peptides in the solid state reveal fibrillar structures of flat morphology that are formed through -sheet mediated self-assembly of the preorganised -turn building blocks.

The effect of PEG crystallization on the morphology of PEG/peptide block copolymers containing amyloid beta-peptide fragments

Ordered nanostructures are observed in the melt and solid state for a series of three peptide/PEG conjugates containing fragments of amyloid beta-peptides. These are conjugated to PEG with (M) over bar (n) = 3 300 g.mol(-1) and a melting temperature T-m = 45-50 degrees C. The morphology at room temperature is examined by AFM and POM. This shows spherulite formation for the weakly fibrillizing KLVFF-PEG sample but fibril formation for FFKLVFF-PEG. The fibrillization tendency of the latter is enhanced by multiple phenylalanine residues. Simultaneous SAXS and WAXS was used to investigate the morphology as a function of temperature. The secondary structure is probed by FTIR.