Rapid acidification and alkylation: Redox analysis of the MHC class I pathway

The technique of rapid acidification and alkylation can be used to characterise the redox status of oxidoreductases, and to determine numbers of free cysteine residues within substrate proteins. We have previously used this method to analyse interacting components of the MHC class I pathway, namely ERp57 and tapasin. Here, we have applied rapid acidification alkylation as a novel approach to analysing the redox status of MHC class I molecules. This analysis of the redox status of the MHC class I molecules HLA-A2 and HLA-B27, which is strongly associated with a group of inflammatory arthritic disorders referred to as Spondyloarthropathies, revealed structural and conformational information. We propose that this assay provides a useful tool in the study of in vivo MHC class I structure. (c) 2008 Elsevier B.V. All rights reserved.

Statistical deconvolution of enthalpic energetic contributions to MHC-peptide binding affinity

Background: MHC Class I molecules present antigenic peptides to cytotoxic T cells, which forms an integral part of the adaptive immune response. Peptides are bound within a groove formed by the MHC heavy chain. Previous approaches to MHC Class I-peptide binding prediction have largely concentrated on the peptide anchor residues located at the P2 and C-terminus positions. Results: A large dataset comprising MHC-peptide structural complexes was created by remodelling pre-determined x-ray crystallographic structures. Static energetic analysis, following energy minimisation, was performed on the dataset in order to characterise interactions between bound peptides and the MHC Class I molecule, partitioning the interactions within the groove into van der Waals, electrostatic and total non-bonded energy contributions. Conclusion: The QSAR techniques of Genetic Function Approximation (GFA) and Genetic Partial Least Squares (G/PLS) algorithms were used to identify key interactions between the two molecules by comparing the calculated energy values with experimentally-determined BL50 data. Although the peptide termini binding interactions help ensure the stability of the MHC Class I-peptide complex, the central region of the peptide is also important in defining the specificity of the interaction. As thermodynamic studies indicate that peptide association and dissociation may be driven entropically, it may be necessary to incorporate entropic contributions into future calculations.

Production of angiotensin-I-converting enzyme (ACE) inhibitory activity in milk fermented with probiotic strains: Effects of calcium, pH and peptides on the ACE-inhibitory activity

Recently, probiotic fermented milk products have raised interest regarding their potential anti-hypertensive activity mainly due to the production of angiotensin-I-converting enzyme (ACE) inhibitory peptides. Ionic calcium released upon milk acidification during fermentation is also known to exert hypotensive activity. Thus, the main aim of this study was to screen probiotic strains for their ability to induce ACE-inhibitory activity upon fermentation of milk. The relationship of ACE-inhibitory activity percentage (ACEi%) with cell growth, pH, degree of hydrolysis and the concentration of ionic calcium released during the fermentation was also investigated. Compared with other lactic acid bacteria, Lactobacillus casei YIT 9029 and Bifidobacterium bifidum MF 20/5 were able to induce strong ACE-inhibitory activity. Furthermore, it was found that the ionic calcium released during milk fermentation could contribute to the ACE-inhibitory activity. These findings will contribute to the development of new probiotic dairy products with anti-hypertensive activity.

Peptides generated ex vivo from serum proteins by tumor-specific exopeptidases are not useful biomarkers in ovarian cancer

BACKGROUND: The serum peptidome may be a valuable source of diagnostic cancer biomarkers. Previous mass spectrometry (MS) studies have suggested that groups of related peptides discriminatory for different cancer types are generated ex vivo from abundant serum proteins by tumor-specific exopeptidases. We tested 2 complementary serum profiling strategies to see if similar peptides could be found that discriminate ovarian cancer from benign cases and healthy controls. METHODS: We subjected identically collected and processed serum samples from healthy volunteers and patients to automated polypeptide extraction on octadecylsilane-coated magnetic beads and separately on ZipTips before MALDI-TOF MS profiling at 2 centers. The 2 platforms were compared and case control profiling data analyzed to find altered MS peak intensities. We tested models built from training datasets for both methods for their ability to classify a blinded test set. RESULTS: Both profiling platforms had CVs of approximately 15% and could be applied for high-throughput analysis of clinical samples. The 2 methods generated overlapping peptide profiles, with some differences in peak intensity in different mass regions. In cross-validation, models from training data gave diagnostic accuracies up to 87% for discriminating malignant ovarian cancer from healthy controls and up to 81% for discriminating malignant from benign samples. Diagnostic accuracies up to 71% (malignant vs healthy) and up to 65% (malignant vs benign) were obtained when the models were validated on the blinded test set. CONCLUSIONS: For ovarian cancer, altered MALDI-TOF MS peptide profiles alone cannot be used for accurate diagnoses.

Factors required for the uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3D(pol)) in vitro

The 5' terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 313 (VTg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3D(pol). To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3D(pol) in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV ere has been identified previously to be within the 5' untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 313 peptides has now been determined, and the role of the FMDV ere (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.

Purification and molecular cloning of antimicrobial peptides from Scots pine seedlings

A novel protocol for rapid and efficient purification of antimicrobial peptides from plant seedlings has been developed. Two peptides with antimicrobial activity, designated p1 and p2, were purified nearly to homogeneity from Scots pine seedlings by a combination of sulfuric acid extraction, ammonium sulfate precipitation, heat-inactivation and ion-exchange chromatography on phosphocellulose. Purified proteins had molecular masses of 11 kDa (p1) and 5.8 kDa (p2) and were identified by mass spectrometry as defensin and lipid-transfer protein, respectively. We demonstrated their growth inhibitory effects against a group of phytopathogenic fungi. Furthermore, we report for the first time molecular cloning and characterization of defensin I cDNA from Scots pine. A cDNA expression library from 7 days Scots pine seedlings was generated and used to isolate a cDNA clone corresponding to Scots pine defensin, termed PsDef1. The full-length coding sequence of PsDef1 is 252 bp in length and has an open reading frame capable to encode a protein of 83 amino residues. The deduced sequence has the typical features of plant defensins, including an endoplasmic reticulum signal sequence of 33 aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5601.6 Da, which correlates well with the results of SDS-PAGE analysis. Finally, the antimicrobial properties of PsDef1 against a panel of fungi and bacteria define it as a member of the morphogenic group of plant defensins. (C) 2009 Elsevier Inc. All rights reserved.

Hydrogelation of self-assembling RGD-based peptides

The self-assembly of tripeptides based on the RGD cell adhesion motif is investigated. Two tripeptides containing the Fmoc [N-(fluorenyl)-9-methoxycarbonyl] aromatic unit were synthesized, Fmoc-RGD and a control peptide containing a scrambled sequence, Fmoc-GRD. The Fmoc is used to control selfassembly via aromatic stacking interactions. The self-assembly and hydrogelation properties of the two Fmoc-tripeptides are compared. Both form well defined amyloid fibrils (as shown by cryo-TEM and SAXS) with b-sheet features in their circular dichroism and FTIR spectra. Both peptides form selfsupporting hydrogels, the dynamic shear modulus of which was measured. Preliminary cell culture experiments reveal that Fmoc-RGD can be used as a support for bovine fibroblasts, but not Fmoc- GRD, consistent with the incorporation of the cell adhesion motif in the former peptide.

Insulin, glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide and insulin-like growth factor I as putative mediators of the hypolipidemic effect of oligofructose in rats

The addition of oligofructose as a dietary fiber decreases the serum concentration and the hepatic release of VLDL-triglycerides in rats. Because glucose, insulin, insulin-like growth factor I (IGF-I) and gut peptides [i.e., glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1)]) are factors involved in the metabolic response to nutrients, this paper analyzes their putative role in the hypolipidemic effect of oligofructose. Male Wistar rats were fed a nonpurified diet with or without 10% oligofructose for 30 d. Glucose, insulin, IGF-I and GIP concentrations were measured in the serum of rats after eating. GIP and GLP-1 contents were also assayed in small intestine and cecal extracts, respectively. A glucose tolerance test was performed in food-deprived rats. Serum insulin level was significantly lower in oligofructose-fed rats both after eating and in the glucose tolerance test, whereas glycemia was lower only in the postprandial state. IGF-I serum level did not differ between groups. GIP concentration was significantly higher in the serum of oligofructose-fed rats. The GLP-1 cecal pool was also significantly higher. In this study, we have shown that cecal proliferation induced by oligofructose leads to an increase in GLP-1 concentration. This latter incretin could be involved in the maintenance of glycemia despite a lower insulinemia in the glucose tolerance test in oligofructose-fed rats. We discuss also the role of hormonal changes in the antilipogenic effect of oligofructose.

Self-assembly of amphiphilic peptides

The self-assembly of amphiphilic peptides is reviewed. The review covers surfactant-like peptides with amphiphilicity arising from the sequence of natural amino acids, and also peptide amphiphiles (PAs) in which lipid chains are attached to hydrophilic peptide sequences containing charged residues. The influence of the secondary structure on the self-assembled structure and vice versa is discussed. For surfactant-like peptides structures including fibrils, nanotubes, micelles and vesicles have been reported. A particularly common motif for PAs is beta-sheet based fibrils, although other structures have been observed. In these structures, the peptide epitope is presented at the surface of the nanostructure, providing remarkable bioactivity. Recent discoveries of potential, and actual, applications of these materials in biomedicine and bionanotechnology are discussed.

Amyloid peptides incorporating a core sequence from the amyloid beta peptide and gamma amino acids: relating bioactivity to self-assembly

A series of heptapeptides comprising the core sequence Ab(16–20), KLVFF, of the amyloid b peptide coupled with paired N-terminal c-amino acids are investigated in terms of cytotoxicity reduction and binding to the full Ab peptide, both pointing to inhibition of fibrillisation for selected compounds. This is related to the self-assembly capacity of the heptapeptides.

Synthesis and incorporation into cyclic peptides of tolan amino acids and their hydrogenated congeners: construction of an array of A–B-loop mimetics of the Cε3 domain of human IgE

The disruption of the human immunolobulin E–high affinity receptor I (IgE–FcεRI) protein–protein interaction (PPI) is a validated strategy for the development of anti asthma therapeutics. Here, we describe the synthesis of an array of conformationally constrained cyclic peptides based on an epitope of the A–B loop within the Cε3 domain of IgE. The peptides contain various tolan (i.e., 1,2-biarylethyne) amino acids and their fully and partially hydrogenated congeners as conformational constraints. Modest antagonist activity (IC50 660 μM) is displayed by the peptide containing a 2,2′-tolan, which is the one predicted by molecular modeling to best mimic the conformation of the native A–B loop epitope in IgE.

Fibrillisation of ring-closed amyloid peptides

Ring-closing olefin metathesis reactions are used to create intramolecularly ring closed peptides or inter-molecularly ring-closed peptide dimers based on a designed amyloid peptide sequence. The uncrosslinked peptide self-assembles into high aspect ratio nanotubes, however ring-closing leads to the formation of fibrillar and twisted/helical ribbon structures.

Control of strand registry by attachment of PEG chains to amyloid peptides influences nanostructure

The self-assembly in aqueous solution of PEG-peptide conjugates comprising a model amyloid peptide sequence FFKLVFF that contains the Ab(16–20) KLVFF motif is investigated. X-ray diffraction reveals different packing motifs dependent on PEG chain length. This is correlated to remarkable differences in self-assembled nanostructures. The control of strand registry points to a subtle interplay between aromatic stacking, electrostatic and amphiphilic interactions.

Production of angiotensin converting enzyme inhibitory peptides from β-lactoglobulin and casein derived peptides: an integrative approach

Angiotensin I-converting enzyme (ACE) inhibition is one of the mechanisms by which reduction in blood pressure is exerted. Whey proteins are a rich source of ACE inhibitory peptides and have shown a blood pressure reduction effect i.e. antihypertensive activity. The aim of this work was to develop a simplified process using a combination of adsorption and microfiltration steps for the production of hydrolysates from whey with high ACE inhibitory activity and potency; the latter was measured as the IC50, which is the peptide concentration required to reduce ACE activity by half. This process integrates the selective separation of β-lactoglobulin and casein derived peptides (CDP) from rennet whey and their hydrolysis, which results in partially pure, less complex hydrolysates with high bioactive potency. Hydrolysis was carried out with protease N ‘Amano’ in a thermostatically controlled membrane reactor operated in a batch mode. By applying the integrative approach it was possible to produce from the same feedstock two different hydrolysates that exhibited high ACE inhibition. One hydrolysate was mainly composed of casein-derived peptides with IC50= 285 μg/mL. In this hydrolysate we identified the well known potent ACE-I and anti-hypertensive tri-peptide Ile-Pro-Pro (IPP) and another novel octa-peptide Gln-Asp-Lys-Thr-Glu-Ile-Pro-Thr (QDKTEIPT). The second hydrolysate was mainly composed of β-lactoglobulin derived peptides with IC50=128 µg/mL. This hydrolysate contained a tetra-peptide (Ile-Ile-Ala-Glu) IIAE as one of the two major peptides. A further advantage to this process is that enzyme activity was substantially increased as enzyme product inhibition was reduced.

Production of novel ACE inhibitory peptides from β-lactoglobulin using Protease N Amano

Potent angiotensin I-converting enzyme (ACE) inhibitory peptide mixtures were obtained from the hydrolysis of β-lactoglobulin (βLg) using Protease N Amano, a food-grade commercial proteolytic preparation. Hydrolysis experiments were carried out for 8 h at two different temperatures and neutral pH. Based on their ACE inhibitory activity, samples of 6 h of digestion were chosen for further analysis. The temperature used for the hydrolysis had a marked influence on the type of peptides produced and their concentration in the hydrolysate. Protease N Amano was found to produce very complex peptide mixtures; however, the partially fractionated hydrolysates had already very potent ACE inhibitory activity. The novel heptapeptide SAPLRVY was isolated and characterised. It corresponded to βLg f(36–42) and had an IC50 value of 8 μm, which is considerably lower than the most potent ACE inhibitory peptides derived from bovine βLg reported so far.