All posts in Raw-Journal Peptide Science

The O-acyl isopeptide method was developed for the efficient preparation of difficult sequence-containing peptide. Furthermore, development of the O-acyl isodipeptide unit for Fmoc chemistry simplified its synthetic procedure by solid-phase peptide synthesis. Here, we report a novel isodipeptide unit for Boc chemistry, and the unit was successfully applied to the synthesis of amyloid β peptide. Combination of Boc chemistry and the isodipeptide unit would be an effective method for the synthesis of many difficult peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

The O-acyl isopeptide method was developed for the efficient preparation of difficult sequence-containing peptides. Furthermore, development of the O-acyl isodipeptide unit for Fmoc chemistry simplified its synthetic procedure by solid-phase peptide synthesis. Here, we report a novel isodipeptide unit for Boc chemistry, and the unit was successfully applied to the synthesis of amyloid β peptide.

Nanoparticles are expected to be applicable for the theranostics as a carrier of the diagnostic and therapeutic agents. Lactosome is a polymeric micelle composed of amphiphilic polydepsipeptide, poly(sarcosine)₆₄-block-poly(l-lactic acid)₃₀, which was found to accumulate in solid tumors through the enhanced permeability and retention effect. However, lactosome was captured by liver on the second administration to a mouse. This phenomenon is called as the accelerated blood clearance phenomenon. On the other hand, peptide-nanosheet composed of amphiphilic polypeptide, poly(sarcosine)₆₀-block-(l-Leu-Aib)₆, where the poly(l-lactic acid) block in lactosome was replaced with the (l-Leu-Aib)₆ block, abolished the accelerated blood clearance phenomenon. The ELISA and in vivo near-infrared fluorescence imaging revealed that peptide-nanosheets did not activate the immune system despite the same hydrophilic block being used. The high surface density of poly(sarcosine) chains on the peptide-nanosheet may be one of the causes of the suppressive immune response. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Polymeric micelles and peptide-nanosheets were prepared with using amphiphilic block polymers having the same hydrophilic poly(sarcosine) block. The former generated anti-poly(sarcosine) IgM as a T-independent antigen, but the latter did not.

Phosphorylation of high mobility group box 1 (HMGB1) is involved in the subcellular translocation of this protein and its subsequent secretion. Two nuclear localization signals (NLSs), NLS1 and NLS2, in this protein regulate its nucleocytoplasmic relocation, and phosphorylation of both NLSs strongly promotes HMGB1 mobilization. However, the phosphorylation properties of serine residues in NLS1 and the kinases involved are not well known. In the present study, we predicted kinases that phosphorylate serine residues in NLS1 and performed an in vitro kinase assay utilizing NLS1-derived phosphopeptides. Among the predicted kinases, protein kinase C phosphorylated Ser⁴⁶ of HMGB1-derived peptides, and a mutagenesis experiment confirmed that phosphorylation at this site could induce the translocation of the N-terminal region of NLS1-containing HMGB1 into the cytosol. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Two nuclear localization signals (NLSs), NLS1 and NLS2, in high mobility group box 1 (HMGB1) regulate its nucleocytoplasmic relocation, and phosphorylation of both NLSs promotes HMGB1 mobilization. In the present study, kinases that phosphorylate serine residues within NLS1 were predicted, and an in vitro kinase assay was performed. Among the predicted kinases, protein kinase C phosphorylated Ser⁴⁶ of HMGB1-derived peptides and a mutagenesis experiment showed that this phosphorylation could induce translocation of the N-terminal region of HMGB1 to the cytosol.

Bicelles are model membrane systems that can be macroscopically oriented in a magnetic field at physiological temperature. The macroscopic orientation of bicelles allows to detect, by means of magnetic resonance spectroscopies, small changes in the order of the bilayer caused by solutes interacting with the membrane. These changes would be hardly detectable in isotropic systems such as vesicles or micelles. The aim of this work is to show that bicelles represent a convenient tool to investigate the behavior of antimicrobial peptides (AMPs) interacting with membranes, using electron paramagnetic resonance (EPR) spectroscopy. We performed the EPR experiments on spin-labeled bicelles using various AMPs of different length, charge, and amphipathicity: alamethicin, trichogin GA IV, magainin 2, HP(2–20), and HPA3. We evaluated the changes in the order parameter of the spin-labeled lipids as a function of the peptide-to-lipid ratio. We show that bicelles labeled at position 5 of the lipid chains are very sensitive to the perturbation induced by the AMPs even at low peptide concentrations. Our study indicates that peptides that are known to disrupt the membrane by different mechanisms (i.e., alamethicin vs magainin 2) show very distinct trends of the order parameter as a function of peptide concentration. Therefore, spin-labeled bicelles proved to be a good system to evaluate the membrane disruption mechanism of new AMPs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

The interaction of bicelles with several antimicrobial peptides, either amphipathic or fully hydrophobic, has been studied with EPR spectroscopy. We show that bicelles are a convenient tool to distinguish peptides with different mechanisms of action on membranes.

Hybrid associates formed between peptide assemblies and fluorophores are attractive mainly because of their unique properties for biomedical applications. Recently, we demonstrated that the production of reactive oxygen species (ROS) by hypericin and their stability in excited states are enhanced upon conjugation with l,l-diphenylalanine microtubes (FF-MNTs). Although the detailed mechanisms responsible for improving the photophysical properties of ROS remain unclear, tentative hypotheses have suggested that the driving force is the growth of overall dipolar moments ascribed either to coupling between aligned H₂O dipoles within the ordered structures or to the organization of hypericin molecules on peptide interfaces. To provide new insights on ROS activity in hypericin/FF-MNTs hybrids and further explore the role of water in this respect, we present results obtained from investigations on the behavior of these complexes organized into different crystalline arrangements. Specifically, we monitored and compared the photophysical performance of hypericin bound to FF-MNTs with peptides organized in both hexagonal (water-rich) and orthorhombic (water-free) symmetries. From a theoretical perspective, we present the results of new molecular dynamics simulations that highlight the distinct hypericin/peptide interaction at the interface of FF-MNTs for the different symmetries. As a conclusion, we propose that although water enhances photophysical properties, the organization induced by peptide structures and the availability of a hydrophobic environment surrounding the hypericin/peptide interface are paramount to optimizing ROS generation. The findings presented here provide useful basic research insights for designing peptide/fluorophore complexes with outstanding technological potential. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

The generation of reactive oxygen species (ROS) by hypericin has been found to be significantly improved upon association with l,l-diphenylalanine microtubes. The photophysical performance of these hybrids was investigated for assemblies organized into both hexagonal (water-rich) and orthorhombic (water-free) symmetries. We have found that organization induced by peptide structures and availability of a hydrophobic micro-environment surrounding the hypericin/peptide interface are paramount to optimizing ROS generation.

Interactions between peptides are relevant from a biomedical point of view, in particular for the role played by their aggregates in different important pathologies, and also because peptide aggregates represent promising scaffolds for innovative materials.

In the present article, the aggregation properties of the homo-peptides formed by α-aminoisobutyric acid (U) residues are discussed. The peptides investigated have chain lengths between six and 15 residues and comprise benzyl and naphthyl groups at the N- and C-termini, respectively. Spectroscopic experiments and molecular dynamics simulations show that the shortest homo-peptide, constituted by six U, does not exhibit any tendency to aggregate under the conditions examined. On the other hand, the homologous peptide with 15 U forms very stable and compact aggregates in 70/30_(v/v) methanol/water solution. Atomic force microscopy images indicate that these aggregates promote formation of long fibrils once they are deposited on a mica surface. The aggregation phenomenon is mainly due to hydrophobic interactions occurring between very stable helical structures, and the aromatic groups in the peptides seem to play a minor role. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

The aggregation properties of Aib homo-peptides have been analyzed by means of computational and experimental techniques. Our findings reveal that the longer homolog of the series, (Aib)₁₅, forms stable aggregates in water/methanol solutions and fibers when dried on mica surface. (Aib)₆, on the contrary, does not show any tendency to aggregate because of its intrinsic higher flexibility in the investigated conditions. During the molecular dynamics simulations (tens of nanoseconds long) of (Aib)₆, both P- and M-helices were populated.

Celiac disease (CD) is an autoimmune mediated disease with complex and multifactorial etiology. Gluten intake triggers a composite immune response involving T-cells and B-cells and leading to the secretion of autoantibodies if a genetic predisposition is present. Untreated CD patients show high levels of circulating autoantibodies directed to different auto-antigens present in the intestinal mucosa. The most important auto-antigen is the endomysial enzyme tissue transglutaminase (tTG). Both IgA and IgG antibody isotypes to tTG are known, but only the IgA antibodies demonstrate the highest disease specificity and thus are considered disease biomarkers. Because the pathogenicity and exact tTG binding properties of these autoantibodies are still unclear, the characterization of tTG antigenic domains is a crucial step in understanding CD onset and the autoimmune pathogenesis. Overlapping peptide libraries can be used for epitope mapping of selected protein portions to determine antigenic fragments contributing to the immunological activity and possibly develop innovative peptide-based tools with high specificity and sensitivity for CD. We performed an epitope mapping study to characterize putative linear auto-antigenic epitopes present in the tTG N-terminal portion (1–230). A library of 23 overlapping peptides spanning tTG(1–230) was generated by Fmoc/tBu solid-phase peptide synthesis and screened by immunoenzymatic assays employing patients' sera. The results indicate that four synthetic peptides, that is, Ac-tTG(1–15)-NH₂, Ac-tTG(41–55)-NH₂, Ac-tTG(51–65)-NH₂, and Ac-tTG(151–165)-NH₂, are recognized by IgA autoantibodies circulating in CD patients' sera. These results offer important insight on the nature of the antigen-antibody interaction. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Mapping of the tissue transglutaminase N-terminal portion (1–230) identified putative linear auto-antigenic epitopes. A library of 23 overlapping peptides was generated by Fmoc/tBu solid-phase peptide synthesis and screened by immunoenzymatic assays. Four peptides are recognized as linear epitopes by IgA autoantibodies circulating in celiac disease patients' sera.

Proteasome is a ‘proteolytic factory’ that constitutes an essential part of the ubiquitin-proteasome pathway. The involvement of proteasome in regulation of all major aspects of cellular physiology makes it an attractive drug target. So far, only inhibitors of the proteasome entered the clinic as anti-cancer drugs. However, proteasome regulators may also be useful for treatment of inflammatory and neurodegenerative diseases. We established in our previous studies that the peptide Tat2, comprising the basic domain of HIV-1 Tat protein: R⁴⁹KKRRQRR⁵⁶, supplemented with Q⁶⁶DPI⁶⁹ fragment, inhibits the 20S proteasome in a noncompetitive manner. Mechanism of Tat2 likely involves allosteric regulation because it competes with the proteasome natural 11S activator for binding to the enzyme noncatalytic subunits. In this study, we performed alanine walking coupled with biological activity measurements and FTIR and CD spectroscopy to dissect contribution of a charge and conformation of Tat2 to its capability to influence peptidase activity of the proteasome. In solution, Tat2 and most of its analogs with a single Ala substitution preferentially adopted a conformation containing PPII/turn structural motifs. Replacing either Asp10 or two or more adjacent Arg/Lys residues induced a random coil conformation, probably by disrupting ionic interactions responsible for stabilization of the peptides ordered structure. The random coil Tat2 analogs lost their capability to activate the latent 20S proteasome. In contrast, inhibitory properties of the peptides more significantly depended on their positive charge. The data provide valuable clues for the future optimization of the Tat2-based proteasome regulators. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Ala scan studies of Tat2 peptide, a noncompetitive inhibitor of human SDS-activated 20S proteasome and a weak activator of the latent 20S, revealed that different peptide features are responsible for its stimulating and inhibitory activity toward the proteasome chymotrypsin-like peptidase. While for stimulating capacity preservation of the PPII/turn conformation seems to play an important role, inhibitory capacity depends rather on the positive charge.