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Inflammation of intestinal tissue in patients affected by celiac disease (CD) originates from the adaptive and innate immune responses elicited by the undigested gliadin fragments through molecular mechanisms not yet completely described. Undigested A-gliadin peptide P31-43 is central to CD pathogenesis, entering enterocytes in vesicular compartments by endocytosis and inducing an innate immune response in CD intestinal mucosa. This study focused on the reasons why P31-43 does not behave as adaptive immunogenic agent. Once obtained by NMR analysis, the three-dimensional model of P31-43 was used to implement a series of in silico experiments aimed to explore the ability of the peptide to interact with HLA-DQ2 and the corresponding receptor onto T cells. Our results show that P31-43 is a poor ligand for DQ2 and/or T-cell receptor. This study was also aimed to investigate, from a structural point of view, the previous experimental findings by which P31-43 is able to enhance the phosphorylation level of the protein ERK2, while some P31-43 Ala-mutants decrease or totally inhibit that process. The molecular models of P31-43, P31-43 P36A, and F37A mutants were used for in silico docking experiments onto the ERK2 structure. The experiments support the hypothesis that P31-43 F37A works as an ERK2 phosphorylation inhibitor because it binds to the ERK2 phosphorylation site. This study reports on the structural properties of so far never NMR characterized gliadin peptides relevant in CD and explores details about their mechanisms of action., (© 2019 European Peptide Society and John Wiley & Sons, Ltd.)

Bone morphogenetic proteins (BMPs) play a key role in bone and cartilage formation. For these properties, BMPs are employed in the field of tissue engineering to induce bone regeneration in damaged tissues. To overcome drawbacks due to the use of entire proteins, synthetic peptides derived from their parent BMPs have come out as promising molecules for biomaterial design. On the structural ground of the experimental BMP-2 receptor complexes reported in the literature, we designed three peptides, reproducing the BMP-2 region responsible for the binding to the type II receptor, ActRIIB. These peptides were characterized by NMR, and the structural features of the peptide-receptor binding interface were highlighted by docking experiments. Peptide-receptor binding affinities were analyzed by means of ELISA and surface plasmon resonance techniques. Furthermore, cellular assays were performed to assess their osteoinductive properties. A chimera peptide, obtained by combining the sequence portions 73-92 and 30-34 of BMP-2, shows the best affinity for ActRIIB in the series and represents a good starting point for the design of new compounds able to reproduce osteogenic properties of the parent BMP-2., (Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.)

Nodal, a member of the TGF-β superfamily, is a potent embryonic morphogen also implicated in tumor progression. As for other TGF-βs, it triggers the signaling functions through the interaction with the extracellular domains of type I and type II serine/threonine kinase receptors and with the co-receptor Cripto. Recently, we reported the molecular models of Nodal in complex with its type I receptors (ALK4 and ALK7) as well as with Cripto, as obtained by homology modeling and docking simulations. From such models, potential binding epitopes have been identified. To validate such hypotheses, a series of mutated Nodal fragments have been synthesized. These peptide analogs encompass residues 44-67 of the Nodal protein, corresponding to the pre-helix loop and the H3 helix, and reproduce the wild-type sequence or bear some modifications to evaluate the hot-spot role of modified residues in the receptor binding. Here, we show the structural characterization in solution by CD and NMR of the Nodal peptides and the measurement of binding affinity toward Cripto by surface plasmon resonance. Data collected by both conformational analyses and binding measurements suggest a role for Y58 of Nodal in the recognition with Cripto and confirm that previously reported for E49 and E50. Surface plasmon resonance binding assays with recombinant proteins show that Nodal interacts in vitro also with ALK7 and ALK4 and preliminary data, generated using the Nodal synthetic fragments, suggest that Y58 of Nodal may also be involved in the recognition with these protein partners., (Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.)

Self-complementary synthetic peptides, composed by 8 and 16 residues, were analyzed by CD, NMR and small angle neutron scattering (SANS) techniques in order to investigate the relevance of charge and hydrophobic interactions in determining their self-assembling properties. All the sequences are potentially able to form fibrils and membranes as they share, with the prototype EAK16, a strictly alternating arrangement of polar and nonpolar residues. We find that 16-mer peptides show higher self-assembling propensities than the 8-mer analogs and that the aggregation processes are favored by salts and neutral pH. Peptide hydrophobic character appears as the most relevant factor in determining self-assembling. Solution conformational analysis, diffusion and SANS measurements all together show that the sequences with a higher self-assemble propensity are distributed, in mild conditions, between light and heavy forms. For some of the systems, the light form is mostly constituted by monomers in a random conformation, while the heavy one is constituted by beta-aggregates. In our study we also verified that sequences designed to adopt extended conformation, when dissolved in alcohol-water mixtures, can easily fold in helix structures. In that media, the prototype of the series appears distributed between helical monomers and beta-aggregates. It is worth noticing that the structural conversion from helical monomer to beta-aggregates, mimics beta-amyloid peptide aggregation mechanisms.

The protein Cripto is the founding member of the extra-cellular EGF-CFC growth factors, which are composed of two adjacent cysteine-rich domains: the EGF-like and the CFC. Members of the EGF-CFC family play key roles in embryonic development and are also implicated in tumourigenesis. Cripto is highly over-expressed in many tumours, while it is poorly detectable in normal tissues. Although both Cripto domains are involved in its tumourigenic activity, the CFC domain appears to play a crucial role. Indeed, through this domain, Cripto interferes with the onco-suppressive activity of Activins, either by blocking the Activin receptor ALK4 or by antagonising proteins of the TGF-beta family. We have undertaken the chemical synthesis and the structural characterisation of human CFC Cripto domain. Using a combined NMR and computational approach, supported by binding studies by SPR, we have investigated the molecular basis of the interaction between h-CFC and ALK4. Binding studies indicate that the synthetic h-CFC interacts with the ALK4 receptor with a K(D) in micro M range, whereas it does not recognise the ActRIIB receptor. The NMR study shows that the h-CFC overall topology is determined by the presence of three disulfide bridges and that residues H120 and W124 are located between the first strand and the first loop with the side chains externally exposed. A model of the CFC-ALK4 complex has also been obtained by molecular docking and shows that all residues indicated by prior mutagenesis studies can contribute to the ALK4-CFC interaction at the protein-protein interface.

Previous studies have indicated that proteolytic activation of pro-hormones and pro-proteins occurs most frequently at the level of basic amino acids arranged in doublets and that the dibasic sites are situated in or next to beta-turns. Investigations utilizing synthetic peptides reproducing the N-terminal processing domain of pro-oxytocin-neurophysin have suggested a close relationship between the secondary structure of the cleavage locus and enzyme recognition, the minimal recognized sequence being the -Pro-Leu-Gly-Gly-Lys-Arg-Ala-Val-Leu- segment of the native precursor. NMR investigations and energy minimization studies have demonstrated that this sequence is organized in two type-II beta-turns involving the -Pro-Leu-Gly-Gly- and -Lys-Arg-Ala-Val- sequences. To further strengthen the above reported hypothesis and to study the role of turn subtypes, a new proline containing cyclic substrate of the processing enzyme, in which the N-terminal side that comes before the Lys-Arg pair is constrained to adopt a type-lI beta-turn, has been synthesized. The presence of a type-II beta-turn structure in this cyclic peptide model has been demonstrated by a combined NMR, CD and FT-IR absorption investigation. A preliminary study shows that PC1 is able to recognize and process our constrained substrate.

The results of a conformational study by nuclear magnetic spectroscopy and computational methods on a series of point-mutated synthetic peptides, containing 14 amino acid residues and mimicking the region containing the Arg-Lys dibasic cleavage site of pro-somatostatin, have confirmed the possible role of a well defined secondary structure in the recognition phenomenon by processing enzymes. The importance of the residues located near the Arg-Lys dibasic site in the C-terminal region of the pro-hormone for the cleavage of the precursor into somatostatin-14 has been confirmed. The present structural analysis indicates the occurrence of two beta-turns in the 4-7 and 11-14 regions, flanking the cleavage site, for all the peptides recognized as substrates by the processing enzyme. Interestingly, in the point-mutated analogue not processed by the enzyme and containing the replacement of proline by alanine in position 5 the first -turn is displaced by one residue and involves the Ala5-Arg8 segment. This observation may explain the lack of recognition by the maturation enzyme.

A series of peptides patterned on the principal neutralizing domain of the HIV-1 envelope glycoprotein gp 120 have been synthesized by solid-phase techniques. Interestingly, in vitro experiments have shown that some of these peptides specifically interact with CD4 and, in particular, that the peptide corresponding to the sequence 307-330 of the HIV-1 MN isolate was able to enhance infection in a dose-specific and not a strain-restricted way. To bypass problems observed in preliminary runs, peptides were synthesized by both Fmoc and Boc chemistry. Comparison of the two strategies has allowed the set up of convenient protocols for the preparation of the target peptides in good yield, and with the high-purity grade needed for biological and physiochemical studies. Since the biological effects were present in the carboxyl-free C-terminal linear peptide but not in the amidated C-terminal analogue, preliminary conformational studies by circular dichroism and nuclear magnetic resonance techniques were also performed in an attempt to correlate these effects with possible contributions of structured conformations as predicted by theoretical calculations. The possibility of a beta-turn structure for the crucial Gly-Pro-Gly-Arg sequence has been confirmed by 2D NMR experiments. Ongoing studies suggest the exploitation of the activating properties of the MN-derived peptides to design a more sensitive and innovative serological test based on the virus itself and not on anti-HIV antibodies, as is the case for the large majority of tests currently in use.

The evaluation of peptide structures in solution is made feasible by the combined use of two-dimensional NMR in the laboratory (NOESY) and rotating frames (ROESY), and by the use of molecular dynamics calculations. The present paper describes how both the NMR method and molecular dynamics calculations were applied to very rigid synthetic bicycle peptides that are analogues of natural amatoxins. The NMR theory, which allows the estimate of interatomic distances between interacting nuclei, is briefly discussed. The experimental data were compared with those of known solid-state structures. Three amatoxin analogues have been examined. Of these, one is biologically active (S-deoxo gamma[R] OH-Ile3-amaninamide) and its structure in the solid state has recently been worked out. The second and third analogues (S-dexo-Ile3-Ala5-amaninamide and S-deoxo-D-Ile3-amaninamide, respectively) are inactive and their solid-state structures are unknown. The data presented confirm the authors previous hypothesis that lack of biological activity of S-deoxo-Ile3-Ala5-amaninamide is due to the masking of the tryptophan ring by the methyl group of L-Ala and not to massive conformational changes of the analogue.

Synthetic peptides reproducing the proteolytic processing site of pro-ocytocin were studied by different spectroscopic techniques, including circular dichroism, Fourier transform infrared absorption, and mono and bidimensional nuclear magnetic resonance, in order to ascertain the possible role of three-dimensional structure in the recognition process by maturation enzymes. Experimental results were compared with energy minimization calculations and suggest that: (i) the region situated on the N-terminus of the Lys-Arg doublet may form a beta-turn; (ii) the sequential organization of the residues participating in the beta-turn determines the privileged relative orientation of the basic amino acid sidechains and the subtype of turn; and (iii) the peptide segment situated on the C-terminal side of the dibasic doublet may assume a helix arrangement. These findings, in spite of the limitations connected to the flexibility of linear peptides, seem to substantiate the hypothesis that structural motifs around the cleavage site could be important for recognition and processing. however, a straightforward correlation between details of the secondary structure and the in vitro reactivity toward a putative convertase is not yet possible.