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High-resolution proton nuclear magnetic resonance (H-1-NMR) spectroscopv (400-600 MHz) was applied to the quantitative analysis of hydroperoxide decomposition products in thermally oxidized oils, Different oils (olive, sunflower and soybean) were heated in a thermostatic bath (180 degrees C for 360 min). Proton NMR quantitative aldehyde analysis (n-alkanals, trans-2-alkenals, 4-hydroxy-trans-2-alkenals, alka-2,4-dienals) of fifteen oil samples (0, 60, 120, 240 and 360 minute heating) was compared with that of total polar compounds through silica column chromatography and of short-chain fatty acids. A good linear relationship (R-2 = 0.94) was found between the unsaturated aldehyde level and the amount of methyl-octanoate as well as between total aldehyde determined by H-1-NMR and total polar compounds (R-2 = 0.92). The same NMR sensitivity obtained at 400 MHz through 3000 scans was reached with only 64 scans for a total acquisition time of 2 min at 600 MHz. The potential use of H-1-NMR as a direct tool to monitor the quality and oxidative degradation in heated and fried oils is discussed.

Cleavage of the HIV envelope glycoprotein gp160 into gp120/gp41 is a prerequisite for virus infectivity. The glycoprotein precursor gp160 undergoes a selective endo-proteolysis at the carboxyl side of a consensus motif consisting of the amino acid sequence Arg508-Glu-Lys510-Arg (site1). The Precursor Convertases (PCs) are proposed enzyme candidates for intracellular processing of the HIV envelope glycoprotein. Furin and PC7, which belong to this endoprotease family, have been shown to correctly cleave gp160. In particular, cleavage occurs selectively at site 1 although other multi-basic sites, which constitute potential substrates for pro-protein convertases, are present. These results suggest that both basic residues and specific secondary structure elements are required for the correct processing of gp160. To determine the role of secondary structure of proteolytic activation, we examined some synthetic peptides reproducing the regions surrounding cleavage site 1 of gp160 HIV-1: 1-19 PTKAKRRVVQREKRAVGIG 505-523 HIV gp160, 1-10 PTKAKRRVVQ 505-514 HIV 1 gp160, 8-19 VVQREKRAVGIG 512-523 HIV-1 gp160. These peptides were analyzed with respect to their ability to be cleaved in vitro by Furin and were structurally characterized by CD and NMR spectroscopy. The kinetic data on cleavage by Furin indicate a clear preference of the enzyme for the larger 1-19 substrate. Furthermore, Furin cleaves this peptide at the processing site 1 REKR-A more efficiently than at the other consensus motif KAKR-R. The experimental data from NMR, here presented, show that in the 1-19 peptide, the cleavage site 1 is likely structured in a loop while the secondary consensus motif is incorporated in a helical structured segment. A model by homology of the three dimensional structure of the catalytic domain of Furin with model substrate 1-19 is here also proposed.

The increasing resistance of fungi to conventional antifungal drugs has prompted worldwide the search for new compounds. In this work, we investigated the antifungal properties of acylated Temporin L derivatives, Pent-1B and Dec-1B, against Candida albicans, including the multidrug-resistant strains. Acylated peptides resulted to be active both on reference and clinical strains with MIC values ranging from 6.5 to 26 µM, and they did not show cytotoxicity on human keratinocytes. In addition, we also observed a synergistic or additive effect with voriconazole for peptides Dec-1B and Pent-1B through the checkerboard assay on voriconazole-resistant Candida strains. Moreover, fluorescence-based assays, NMR spectroscopy, and confocal microscopy elucidated a potential membrane-active mechanism, consisting of an initial electrostatic interaction of acylated peptides with fungal membrane, followed by aggregation and insertion into the lipid bilayer and causing membrane perturbation probably through a carpeting effect.

Worldwide, over 20 million patients suffer from bone disorders annually. Bone scaffolds are designed to integrate into host tissue without causing adverse reactions. Recently, chitosan, an easily available natural polymer, has been considered a suitable scaffold for bone tissue growth as it is a biocompatible, biodegradable, and non-toxic material with antimicrobial activity and osteoinductive capacity. In this work, chitosan was covalently and selectively biofunctionalized with two suitably designed bioactive synthetic peptides: a Vitronectin sequence (HVP) and a BMP-2 peptide (GBMP1a). Nuclear magnetic resonance (NMR), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FT-IR) investigations highlighted the presence of the peptides grafted to chitosan (named Chit-HVP and Chit-GBMP1a). Chit-HVP and Chit-GBMP1a porous scaffolds promoted human osteoblasts adhesion, proliferation, calcium deposition, and gene expression of three crucial osteoblast proteins. In particular, Chit-HVP highly promoted adhesion and proliferation of osteoblasts, while Chit-GBMP1a guided cell differentiation towards osteoblastic phenotype.

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.

Antimicrobial peptides (AMPs) represent a skilled class of new antibiotics, due to their broad range of activity, rapid killing, and low bacterial resistance. Many efforts have been made to discover AMPs with improved performances, i.e., high antimicrobial activity, low cytotoxicity against human cells, stability against proteolytic degradation, and low costs of production. In the design of new AMPs, several physicochemical features, such as hydrophobicity, net positive charge, propensity to assume amphipathic conformation, and self-assembling properties, must be considered. Starting from the sequence of the dodecapeptide 1018-K6, we designed a new 10-aminoacid peptide, namely RiLK1, which is highly effective against both fungi and Gram-positive and -negative bacteria at low micromolar concentrations without causing human cell cytotoxicity. In order to find the structural reasons explaining the improved performance of RiLK1 versus 1018-K6, a comparative analysis of the two peptides was carried out with a combination of CD, NMR, and fluorescence spectroscopies, while their self-assembling properties were analyzed by optical and atomic force microscopies. Interestingly, the different spectroscopic and microscopic profiles exhibited by the two peptides, including the propensity of RiLK1 to adopt helix arrangements in contrast to 1018-K6, could explain the improved bactericidal, antifungal, and anti-biofilm activities shown by the new peptide against a panel of food pathogens.

GADD45&beta, /MKK7 complex is a non-redundant, cancer cell-restricted survival module downstream of the NF-kB survival pathway, and it has a pathogenically critical role in multiple myeloma, an incurable malignancy of plasma cells. The first-in-class GADD45&beta, /MKK7 inhibitor DTP3 effectively kills MM cells expressing its molecular target, both in vitro and in vivo, by inducing MKK7/JNK-dependent apoptosis with no apparent toxicity to normal cells. DTP3 combines favorable drug-like properties, with on-target-specific pharmacology, resulting in a safe and cancer-selective therapeutic effect, however, its mode of action is only partially understood. In this work, we have investigated the molecular determinants underlying the MKK7 interaction with DTP3 by combining computational, NMR, and spectroscopic methods. Data gathered by fluorescence quenching and computational approaches consistently indicate that the N-terminal region of MKK7 is the optimal binding site explored by DTP3. These findings further the understanding of the selective mode of action of GADD45&beta, /MKK7 inhibitors and inform potential mechanisms of drug resistance. Notably, upon validation of the safety and efficacy of DTP3 in human trials, our results could also facilitate the development of novel DTP3-like therapeutics with improved bioavailability or the capacity to bypass drug resistance.

Gluten fragments released in gut of celiac individuals activate the innate or adaptive immune systems. The molecular mechanisms associated with the adaptive response involve a series of immunodominant gluten peptides which are mainly recognized by human leucocyte antigen (HLA)-DQ2.5 and HLA-DQ8. Other peptides, such as A-gliadin P31&ndash, 43, are not recognized by HLA and trigger innate responses by several routes not yet well detailed. Among the gluten fragments known to be active in Celiac disease, here we focus on the properties of all gluten peptides with known tri-dimensional structure either those locked into HLA-DQ complexes whose crystals were X-ray analyzed or characterized in solution as free forms. The aim of this work was to find the structural reasons why some gluten peptides prompt the adaptive immune systems while others do not, by apparently involving just the innate immune routes. We propose that P31&ndash, 43 is a non-adaptive prompter because it is not a good ligand for HLA-DQ. Even sharing a similar ability to adopt polyproline II structure with the adaptive ones, the way in which the proline residues are located along the sequence disfavors a productive P31&ndash, 43-HLA-DQ binding.

Recombinant antibodies fragments in several new formats are routinely investigated and used in diagnostic and therapeutic applications as anti-cancers molecules. New antibody formats are generated to compensate the need for multispecificity and site-specific introduction of fluorescent dyes, cytotoxic payloads or for generating semisynthetic multimeric molecules. Fabs of trastuzumab bearing transglutaminase (MTG) reactive sites were generated by periplasmic expression in E. coli and purified. Multimeric Fabs were generated by either disulfide bridge formation or by using MTG-sensitive peptide linkers. Binding to receptor was assessed by ELISA and SPR methods. Internalization and growth inhibition assays were performed on BT-474 and SKBR3 Her2+ cells. Fabs were successfully produced and dimerized or trimerized using MTG and suitably designed peptide linkers. Site-specific derivatizations with fluorophores were similarly achieved. The monomeric, dimeric and trimeric variants bind the receptor with affinities similar or superior to the full antibody. Fab and Fab2 are rapidly internalized in Her2+ cells and exhibit growth inhibition abilities similar to the full antibody. Altogether, the data show that the recombinant Fabs can be produced in E. coli and converted into multimeric variants by MTG-based bioconjugation. Similar approaches are extendable to the introduction of cytotoxic payloads for the generation of novel Antibody Drug Conjugates.

Hydrogels produced by self-assembling peptides are intrinsically biocompatible and thus appropriate for many biomedical purposes. Their application field may be even made wider by reducing the softness and improving the hydrogel mechanical properties through cross-linking treatments. To this aim, modifications of EAK16-II sequence by including Cys residues in its sequence were here investigated in order to obtain hydrogels cross-linkable through a disulfide bridge. Two sequences, namely, C-EAK and C-EAK-C, that contain Cys residues at the N-terminus or at both ends were characterized. Fiber-forming abilities and biological and dynamic mechanical properties were explored before and after the oxidative treatment. In particular, the oxidized version of C-EAK presents a good cell viability and sustains osteoblast proliferation. Furthermore, molecular dynamics (MD) simulations on monomeric and assembled forms of the peptides were performed. MD simulations explained how a specific Cys functionalization was better than the other one. In particular, the results suggested that EAK16-II functionalization with a single Cys residue, instead of two, together with biocompatible cross-linking may be considered an intriguing strategy to obtain a support with better dynamic mechanical properties and biological performances.

The ability of many proteins to fold into well-defined structures has been traditionally considered a prerequisite for fulfilling their functions. Protein folding is also regarded as a valuable loophole to escape uncontrolled and harmful aggregations. Here we show that the PBX-regulating protein-1 (PREP1), an important homeodomain transcription factor involved in cell growth and differentiation during embryogenesis, is endowed with an uncommon thermostability. Indeed, circular dichroism analyses indicate that it retains most of its secondary structure at very high temperatures. These findings have important implications for PREP1 functions since it is a stabilizing factor of its partner PBX1. Predictive analyses suggest that the observed PREP1 thermostability could be related to the presence of aggregation-prone regions. Interestingly, synthetic peptides corresponding to these regions exhibit a remarkable propensity to form toxic β-rich amyloid-like aggregates in physiological conditions. On this basis, we suggest that PREP1 stability is an effective way to prevent or limit the formation of harmful aggregates. Notably, one of these PREP1 fragments (residues 117-132) is able to reversibly switch from α-helical to β-rich states depending on the environmental conditions. The chameleon conformational behavior of this peptide makes it an ideal system to study this intriguing and widespread structural transition.

Synthetic peptides of different size, reproducing the proteolytic processing site of proocytocin, were studied by CD under several experimental conditions in order to ascertain the ability of different solvents to stabilize secondary structural motifs, such as alpha-helix tracts and beta-turns. A combination of deconvolution methods and empirical calculations subtracting the contributions due to unordered structures from the spectra suggests that in solution (a) mainly two distinct families of ordered conformers containing structurally different beta-turns are present, (b) the relative stability of the different conformers depends from the nature of the solvent, and (c) in the case of the larger peptides, a population containing an alpha-helical conformation is also present. From the biological point of view the presence of at least two families of ordered conformers could be in line with current theories assuming that the catalytic effect of the receptor microenvironment may be determinant in shifting the equilibrium toward the active conformation.