The mitochondrial targeting function of randomly generated peptide sequences correlates with predicted helical amphiphilicity.

A pool of oligonucleotides encoding a start methionine and nine random amino acids was inserted at the 5'-end of the gene for the yeast cytochrome oxidase subunit IV lacking its own mitochondrial targeting sequence. Approximately one-quarter of the randomly generated sequences targeted subunit IV to its correct intramitochondrial location in vivo. Sequence analysis of 89 randomly generated sequences showed that their efficiencies as mitochondrial targeting signals correlated with the potential to fold into an amphiphilic alpha-helix. Functional targeting sequences were enriched in arginine and isoleucine residues but contained few aspartate, glutamate, and proline residues. Nonfunctional sequences predicted to have significant helical amphiphilicity often had at least one acidic or multiple helix-breaking residues that would be expected to interfere with targeting functioning. These results support the hypothesis that the signal for targeting a protein into the mitochondrial matrix is usually a positively charged amphiphilic helix.

Solution self-assembly of hybrid block copolymers containing poly(ethylene glycol) and amphiphilic beta-strand peptide sequences

The self-assembly in aqueous solution of hybrid block copolymers consisting of amphiphilic β-strand peptide sequences flanked by one or two PEG chains was investigated by means of circular dichroism spectroscopy, small-angle X-ray scattering, and transmission electron microscopy. In comparison with the native peptide sequence, it was found that the peptide secondary structure was stabilized against pH variation in the di-and tri-block copolymers with PEG. Small-angle X-ray scattering indicated the presence of fibrillar structures, the dimensions of which are comparable to the estimated width of a β-strand (with terminal PEG chains in the case of the copolymers). Transmission electron microscopy on selectively stained and dried specimens shows directly the presence of fibrils. It is proposed that these fibrils result from the hierarchical self-assembly of peptide β-strands into helical tapes, which then stack into fibrils.

Macrocycles mimic the extended peptide conformation recognized by aspartic, serine, cysteine and metallo proteases

It has been previously demonstrated that aspartic, serine, metallo and cysteine proteases bind to their inhibitors and substrate analogues in a single conformation, the saw-tooth or extended beta-strand. Consequently a generic approach to the development of protease inhibitors is the use of constraints that conformationally restrict putative inhibitor molecules to an extended form. In this way the inhibitor is pre-organized for binding to a protease and does not need to rearrange its structure. One constraining device that has proven to be effective for such pre-organization is macrocyclization. This article illustrates the general principle that macrocycles, especially those composed of 3-4 amino acids and usually 13-17 ring atoms, can effectively mimic the extended conformation of short peptide sequences. Such structure-stabilising macrocycles are stable to degradation by proteases, valuable components of potent protease inhibitors, and in many cases they are also bioavailable.

CD8+ T-cell response to NY-ESO-1: relative antigenicity and in vitro immunogenicity of natural and analogue sequences.

We have shown previously that HLA-A*0201 melanoma patients can frequently develop a CTL response to the cancer testis antigen NY-ESO-1. In the present study, we have analyzed in detail the relative antigenicity and in vitro immunogenicity of natural and modified NY-ESO-1 peptide sequences. The results of this analysis revealed that, although suboptimal for binding to the HLA-A*0201 molecule, peptide NY-ESO-1 157-165 is, among natural sequences, very efficiently recognized by specific CTL clones derived from three melanoma patients. In contrast, peptides NY-ESO-1 157-167 and NY-ESO-1 155-163, which bind very strongly to HLA-A*0201, are recognized less efficiently. In agreement with previous data, substitution of peptide NY-ESO-1 157-165 COOH-terminal C with various other amino acids resulted in a significantly increased binding to HLA-A*0201 molecules as well as in an increased CTL recognition, although variable at the clonal level. Among natural peptides, NY-ESO-1 157-165 and NY-ESO-1 157-167 exhibited good in vitro immunogenicity, whereas peptide NY-ESO-1 155-163 was poorly immunogenic. The fine specificity of interaction between peptide NY-ESO-1 C165A, HLA-A*0201, and T-cell receptor was analyzed at the molecular level using a series of variant peptides containing single alanine substitutions. The findings reported here have significant implications for the formulation of NY-ESO-1-based vaccines as well as for the monitoring of either natural or vaccine-induced NY-ESO-1-specific CTL responses in cancer patients.

Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues.

The Melan-A/MART-1 gene, which is expressed by normal melanocytes as well as by most fresh melanoma samples and melanoma cell lines, codes for Ags recognized by tumor-reactive CTL. HLA-A*0201-restricted Melan-A-specific CTL recognize primarily the Melan-A(27-35) (AAGIGILTV) and the Melan-A(26-35) (EAAGIGILTV) peptides. The sequences of these two peptides are not necessarily optimal as far as binding to HLA-A*0201 is concerned, since both lack one of the dominant anchor amino acid residues (leucine or methionine) at position 2. In this study we introduced single amino acid substitutions in either one of the two natural peptide sequences with the aim of improving peptide binding to HLA-A*0201 and/or recognition by specific CTL. Surprisingly, analogues of the Melan-A(27-35) peptide, which bound more efficiently than the natural nonapeptide to HLA-A*0201, were poorly recognized by tumor-reactive CTL. In contrast, among the Melan-A(26-35) peptide analogues tested, the peptide ELAGIGILTV was not only able to display stable binding to HLA-A2.1 but was also recognized more efficiently than the natural peptide by two short-term cultured tumor-infiltrated lymph node cell cultures as well as by five of five tumor-reactive CTL clones. Moreover, in vitro generation of tumor-reactive CTL by stimulation of PBMC from HLA-A*0201 melanoma patients with this particular peptide analogue was much more efficient than that observed with either one of the two natural peptides. These results suggest that the Melan-A(26-35) peptide analogue ELAGIGILTV may be more immunogenic than the natural peptides in HLA-A*0201 melanoma patients and should thus be considered as a candidate for future peptide-based vaccine trials.

Activation of human melanoma reactive CD8+ T cells by vaccination with an immunogenic peptide analog derived from Melan-A/melanoma antigen recognized by T cells-1.

PURPOSE: As compared with natural tumor peptide sequences, carefully selected analog peptides may be more immunogenic and thus better suited for vaccination. However, T cells in vivo activated by such altered analog peptides may not necessarily be tumor specific because sequence and structure of peptide analogs differ from corresponding natural peptides. EXPERIMENTAL DESIGN: Three melanoma patients were immunized with a Melan-A peptide analog that binds more strongly to HLA-A*0201 and is more immunogenic than the natural sequence. This peptide was injected together with a saponin-based adjuvant, followed by surgical removal of lymph node(s) draining the site of vaccination. RESULTS: Ex vivo analysis of vaccine site draining lymph nodes revealed antigen-specific CD8+ T cells, which had differentiated to memory cells. In vitro, these cells showed accelerated proliferation upon peptide stimulation. Nearly all (16 of 17) of Melan-A-specific CD8+ T-cell clones generated from these lymph nodes efficiently killed melanoma cells. CONCLUSIONS: Patient immunization with the analog peptide leads to in vivo activation of T cells that were specific for the natural tumor antigen, demonstrating the usefulness of the analog peptide for melanoma immunotherapy.

Peptide-decorated chitosan derivatives enhance fibroblast adhesion and proliferation in wound healing.

RGD peptide sequences are known to regulate cellular activities by interacting with α5β1, αvβ5 and αvβ3 integrin, which contributes to the wound healing process. In this study, RGDC peptide was immobilized onto chitosan derivative 1,6-diaminohexane-O-carboxymethyl-N,N,N-trimethyl chitosan (DAH-CMTMC) to display RGDC-promoting adhesion for enhanced wound healing. The efficiency of N-methylation, O-carboxymethylation and spacer grafting was quantitatively and qualitatively analyzed by (1)H NMR and FTIR, yielding 0.38 degree of substitution for N-methylation and >0.85 for O-carboxymethylation. The glass transition temperatures for chitosan derivatives were also studied. Peptide immobilization was achieved through sulfhydryl groups using sulfosuccinimidyl (4-iodoacetyl)amino-benzoate (sulfo-SIAB method). RGDC immobilized peptide onto DAH-CMTMC was found to be about 15.3μg/mg of chitosan derivative by amino acid analysis (AAA). The significant increase of human dermal fibroblast (HDF) viability in vitro over 7 days suggests that RGDC-functionalized chitosan may lead to enhanced wound healing (viability >140%). Moreover, bio-adhesion and proliferation assays confirmed that coatings of RGDC-functionalized chitosan derivatives exhibit in vitro wound healing properties by enhancing fibroblast proliferation and adhesion. These results showed that RGDC peptide-functionalized chitosan provides an optimal environment for fibroblast adhesion and proliferation.

Self assembly of a model amphiphilic phenylalanine peptide/polyethylene glycol block copolymer in aqueous solution

There has been great interest recently in peptide amphiphiles and block copolymers containing biomimetic peptide sequences due to applications in bionanotechnology. We investigate the self-assembly of the peptide-PEG amphiphile FFFF-PEG5000 containing the hydrophobic sequence of four phenylalanine residues conjugated to PEG of molar mass 5000. This serves as a simple model peptide amphiphile. At very low concentration, association of hydrophobic aromatic phenylalanine residues occurs, as revealed by circular dichroism and UV/vis fluorescence experiments. A critical aggregation concentration associated with the formation of hydrophobic domains is determined through pyrene fluorescence assays. At higher concentration, defined beta-sheets develop as revealed by FTIR spectroscopy and X-ray diffraction. Transmission electron microscopy reveals self-assembled straight fibril structures. These are much shorter than those observed for amyloid peptides, the finite length may be set by the end cap energy due to the hydrophobicity of phenylalanine. The combination of these techniques points to different aggregation processes depending on concentration. Hydrophobic association into irregular aggregates occurs at low concentration, well-developed beta-sheets only developing at higher concentration. Drying of FFFF-PEG5000 solutions leads to crystallization of PEG, as confirmed by polarized optical microscopy (POM), FTIR and X-ray diffraction (XRD). PEG crystallization does not disrupt local beta-sheet structure (as indicated by FTIR and XRD). However on longer lengthscales the beta-sheet fibrillar structure is perturbed because spheruilites from PEG crystallization are observed by POM. (C) 2009 Elsevier B.V. All rights reserved.

Peptide mediated formation of hierarchically organized solution and solid state polymer nanostructures

Biologically-inspired peptide sequences have been explored as auxiliaries to mediate self-assembly of synthetic macromolecules into hierarchically organized solution and solid state nanostructures. Peptide sequences inspired by the coiled coil motif and "switch" peptides, which can adopt both amphiphilic alpha-helical and beta-strand conformations, were conjugated to poly(ethylene glycol) (PEG). The solution and solid state self-assembly of these materials was investigated using a variety of spectroscopic, scattering and microscopic techniques. These experiments revealed that the folding and organization properties of the peptide sequences are retained upon conjugation of PEG and that they provide the driving force for the formation of the different nanoscale structures which were observed. The possibility of using defined peptide sequences to direct structure formation of synthetic polymers together with the potential of peptide sequences to induce a specific biological response offers interesting prospects for the development of novel self-assembled and biologically active materials.

Altering peptide fibrillization by polymer conjugation

A strategy is presented that exploits the ability of synthetic polymers of different nature to disturb the strong selfassembly capabilities of amyloid based β-sheet forming peptides. Following a convergent approach, the peptides of interest were synthesized via solid-phase peptide synthesis (SPPS) and the polymers via reversible addition−fragmentation chain transfer (RAFT) polymerization, followed by a copper(I) catalyzed azide− alkyne cycloaddition (CuAAC) to generate the desired peptide− polymer conjugates. This study focuses on a modified version of the core sequence of the β-amyloid peptide (Aβ), Aβ(16−20) (KLVFF). The influence of attaching short poly(Nisopropylacrylamide) and poly(hydroxyethylacrylate) to the peptide sequences on the self-assembly properties of the hybrid materials were studied via infrared spectroscopy, TEM, circular dichroism and SAXS. The findings indicate that attaching these polymers disturbs the strong self-assembly properties of the biomolecules to a certain degree and permits to influence the aggregation of the peptides based on their β-sheets forming abilities. This study presents an innovative route toward targeted and controlled assembly of amyloid-like fibers to drive the formation of polymeric nanomaterials.

PEG−peptide conjugates

The remarkable diversity of the self-assembly behavior of PEG−peptides is reviewed, including self-assemblies formed by PEG−peptides with β-sheet and α-helical (coiled-coil) peptide sequences. The modes of self-assembly in solution and in the solid state are discussed. Additionally, applications in bionanotechnology and synthetic materials science are summarized.

Fmoc-POAC: [(9-fluorenylmethyloxycarbonyl)-2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid]: A novel protected spin labeled beta-amino acid for peptide and protein chemistry

The stable free radical 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) is the only spin labeled amino acid that has been used to date to successfully label peptide sequences for structural studies. However, severe difficulty in coupling the subsequent amino acid has been the most serious shortcoming of this paramagnetic marker. This problem stems from the low nucleophilicity of TOAC's amine group towards the acylation reaction during peptide chain elongation. The present report introduces the alternative beta -amino acid 2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid (POAC), potentially useful in peptide and protein chemistry. Investigations aimed at addressing the stereochemistry of this cyclic molecule through X-ray diffraction measurements of crystalline and bulk samples revealed that it consists only of the trans conformer. The 9-fluorenylmethyloxyearbonyl group (Fmoc) was chosen for temporary protection of the POAC amine function, allowing insertion of the probe at any position in a peptide sequence. The vasoactive octapeptide angiotensin II (AII, DRVYIHPF) was synthesized by replacing Pro(7) with POAC. The reaction of Fmoc-POAC with the peptidyl-resin occurred smoothly, and the coupling of the subsequent amino acid showed a much faster reaction when compared with TOAC. POAC(7)-AII was obtained in good yield, demonstrating that, in addition to TOAC, POAC is a convenient amino acid for the synthesis of spin labeled peptide analogues. The present findings open the possibility of a wide range of chemical and biological applications for this novel beta -amino acid derivative, including structural investigations involving its differentiated bend-inducing characteristics.

Alanine is helix-stabilizing in both template-nucleated and standard peptide helices

Alanine-based peptides of defined sequence and length show measurable helix contents, allowing them to be used as a model system both for analyzing the mechanism of helix formation and for investigating the contributions of side-chain interactions to protein stability. Extensive characterization of many peptide sequences with varying amino acid contents indicates that the favorable helicity of alanine-based peptides can be attributed to the large helix-stabilizing propensity of alanine. Based on their analysis of alanine-rich sequences N-terminally linked to a synthetic helix-inducing template, Kemp and coworkers [Kemp, D. S., Boyd, J. G. & Muendel, C. C. (1991) Nature (London) 352, 451–454; Kemp, D. S., Oslick, S. L. & Allen, T. J. (1996) J. Am. Chem. Soc. 118, 4249–4255] argue that alanine is helix-indifferent, however, and that the favorable helix contents of alanine-based peptides must have some other explanation. Here, we show that the helix contents of template-nucleated sequences are influenced strongly by properties of the template–helix junction. A model in which the helix propensities of residues at the template–peptide junction are treated separately brings the results from alanine-based peptides and template-nucleated helices into agreement. The resulting model provides a physically plausible resolution of the discrepancies between the two systems and allows the helix contents of both template-nucleated and standard peptide helices to be predicted by using a single set of helix propensities. Helix formation in both standard peptides and template–peptide conjugates can be attributed to the large intrinsic helix-forming tendency of alanine.

The identification of CD4+ T cell epitopes with dedicated synthetic peptide libraries

For a large number of T cell-mediated immunopathologies, the disease-related antigens are not yet identified. Identification of T cell epitopes is of crucial importance for the development of immune-intervention strategies. We show that CD4+ T cell epitopes can be defined by using a new system for synthesis and screening of synthetic peptide libraries. These libraries are designed to bind to the HLA class II restriction molecule of the CD4+ T cell clone of interest. The screening is based on three selection rounds using partial release of 14-mer peptides from synthesis beads and subsequent sequencing of the remaining peptide attached to the bead. With this approach, two peptides were identified that stimulate the β cell-reactive CD4+ T cell clone 1c10, which was isolated from a newly diagnosed insulin-dependent diabetes mellitus patient. After performing amino acid-substitution studies and protein database searches, a Haemophilus influenzae TonB-derived peptide was identified that stimulates clone 1c10. The relevance of this finding for the pathogenesis of insulin-dependent diabetes mellitus is currently under investigation. We conclude that this system is capable of determining epitopes for (autoreactive) CD4+ T cell clones with previously unknown peptide specificity. This offers the possibility to define (auto)antigens by searching protein databases and/or to induce tolerance by using the peptide sequences identified. In addition the peptides might be used as leads to develop T cell receptor antagonists or anergy-inducing compounds.

Two-hybrid system as a model to study the interaction of beta-amyloid peptide monomers.

The kinetics of amyloid fibril formation by beta-amyloid peptide (Abeta) are typical of a nucleation-dependent polymerization mechanism. This type of mechanism suggests that the study of the interaction of Abeta with itself can provide some valuable insights into Alzheimer disease amyloidosis. Interaction of Abeta with itself was explored with the yeast two-hybrid system. Fusion proteins were created by linking the Abeta fragment to a LexA DNA-binding domain (bait) and also to a B42 transactivation domain (prey). Protein-protein interactions were measured by expression of these fusion proteins in Saccharomyces cerevisiae harboring lacZ (beta-galactosidase) and LEU2 (leucine utilization) genes under the control of LexA-dependent operators. This approach suggests that the Abeta molecule is capable of interacting with itself in vivo in the yeast cell nucleus. LexA protein fused to the Drosophila protein bicoid (LexA-bicoid) failed to interact with the B42 fragment fused to Abeta, indicating that the observed Abeta-Abeta interaction was specific. Specificity was further shown by the finding that no significant interaction was observed in yeast expressing LexA-Abeta bait when the B42 transactivation domain was fused to an Abeta fragment with Phe-Phe at residues 19 and 20 replaced by Thr-Thr (AbetaTT), a finding that is consistent with in vitro observations made by others. Moreover, when a peptide fragment bearing this substitution was mixed with native Abeta-(1-40), it inhibited formation of fibrils in vitro as examined by electron microscopy. The findings presented in this paper suggest that the two-hybrid system can be used to study the interaction of Abeta monomers and to define the peptide sequences that may be important in nucleation-dependent aggregation.