Formation of fibrillar structures through self-assembly of designed peptide turns

Three tripeptides Boc-Phe-Aib-Val-OMe (1), Boc-Leu-Aib-p-NA-NO2 (2) and Boc-Pro-Aib-m-NA-NO2 (3) (Aib: alpha-aminoisobutyric acid; p- and m-NA: para- and meta-nitroaniline) have been designed by incorporating aromatic rings to study the self-assembly and fibril formation. Single crystal X-ray diffraction studies show that all the peptides adopt turn-like structures that are self-assembled through intermolecular hydrogen bonds and van der Waals interactions to create layers of beta-sheets. Solvent dependent NMR titration and CD studies show that the turn structures of the peptides also exist in the solution phase. The field emission scanning electron microscopic (FE-SEM) images of the peptides in the solid state reveal fibrillar structures of flat morphology that are formed through beta-sheet mediated self-assembly of the preorganized turn building blocks.

Overlapping double turn conformations adopted by tetrapeptides containing non-coded alpha-Amino Isobutyric Acid (AIB) and formation of tape-like structures through supramolecular helix mediated self-assembly

Single crystal X-ray diffraction studies and solvent dependent H-1 NMR titrations reveal that a set of four tetrapeptides with general formula Boc-Xx(1)-Aib(2)-Yy(3)-Zz(4)-OMe, where Xx, Yy and Zz are coded L- amino acids, adopt equivalent conformations that can be described as overlapping double turn conformations stabilized by two 4 -> 1 intramolecular hydrogen bonds between Yy(3)-NH and Boc C=O and Zz(4)-NH and Xx(1)C=O. In the crystalline state, the double turn structures are packed in head-to-tail fashion through intermolecular hydrogen bonds to create supramolecular helical structures. Field emission scanning electron microscopic (FE-SEM) images of the tetrapeptides in the solid state reveal that they can form flat tape-like structures. The results establish that synthetic Aib containing supramolecular helices can form highly ordered self-aggregated amyloid plaque like human amylin.

Soy isoflavones increase preprandial peptide YY (PYY), but have no effect on ghrelin and body weight in healthy postmenopausal women

Background: Soy isoflavones show structural and functional similarities to estradiol. Available data indicate that estradiol and estradiol-like components may interact with gut "satiety hormones" such as peptide YY (PYY) and ghrelin, and thus influence body weight. In a randomized, double-blind, placebo-controlled, cross-over trial with 34 healthy postmenopausal women (59 ± 6 years, BMI: 24.7 ± 2.8 kg/m2), isoflavone-enriched cereal bars (50 mg isoflavones/day; genistein to daidzein ratio 2:1) or non-isoflavone-enriched control bars were consumed for 8 weeks (wash-out period: 8-weeks). Seventeen of the subjects were classified as equol producers. Plasma concentrations of ghrelin and PYY, as well as energy intake and body weight were measured at baseline and after four and eight weeks of each intervention arm. Results: Body weight increased in both treatment periods (isoflavone: 0.40 ± 0.94 kg, P < 0.001; placebo: 0.66 ± 0.87 kg, P = 0.018), with no significant difference between treatments. No significant differences in energy intake were observed (P = 0.634). PYY significantly increased during isoflavone treatment (51 ± 2 pmol/L vs. 55 ± 2 pmol/L), but not during placebo (52 ± 3 pmol/L vs. 50 ± 2 pmol/L), (P = 0.010 for treatment differences, independent of equol production). Baseline plasma ghrelin was significantly lower in equol producers (110 ± 16 pmol/L) than in equol non-producers (162 ± 17 pmol/L; P = 0.025). Conclusion: Soy isoflavone supplementation for eight weeks did not significantly reduce energy intake or body weight, even though plasma PYY increased during isoflavone treatment. Ghrelin remained unaffected by isoflavone treatment. A larger and more rigorous appetite experiment might detect smaller differences in energy intake after isoflavone consumption. However, the results of the present study do not indicate that increased PYY has a major role in the regulation of body weight, at least in healthy postmenopausal women.

Self-assembly of a designed amyloid peptide containing the functional thienylalanine unit

The self-assembly of a peptide based on a sequence from the amyloid beta peptide but incorporating the non-natural amino acid beta-2-thienylalanine (2-Thi) has been investigated in aqueous and methanol solutions. The peptide AAKLVFF was used as a design motif, replacing the phenylalanine residues (F) with 2-Thi units to yield (2-Thi)(2-Thi)VLKAA. The 2-Thi residues are expected to confer interesting electronic properties due to charge delocalization and pi-stacking. The peptide is shown to form beta-sheet-rich amyloid fibrils with a twisted morphology, in both water and methanol solutions at sufficiently high concentration. The formation of a self-assembling hydrogel is observed at high concentration. Detailed molecular modeling using molecular dynamics methods was performed using NOE constraints provided by 2D-NMR experiments. The conformational and charge properties of 2-Thi were modeled using quantum mechanical methods, and found to be similar to those previously reported for the beta-3-thienylalanine analogue. The molecular dynamics simulations reveal well-defined folded structures (turn-like) in dilute aqueous solution, driven by self-assembly of the hydrophobic aromatic units, with charged lysine groups exposed to water.

Highly asymmetric phase diagram of a poly(1,2-octylene oxide)-poly(ethylene oxide) diblock copolymer system comprising a brush-like poly(1,2-octylene oxide) block

The phase diagram of a series of poly(1,2-octylene oxide)-poly(ethylene oxide) (POO-PEO) diblock copolymers is determined by small-angle X-ray scattering. The Flory-Huggins interaction parameter was measured by small-angle neutron scattering. The phase diagram is highly asymmetric due to large conformational asymmetry that results from the hexyl side chains in the POO block. Non-lamellar phases (hexagonal and gyroid) are observed near f(PEO) = 0.5, and the lamellar phase is observed for f(PEO) >= 0.5.

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.

Localization of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in human gastrointestinal tract

Calcitonin gene-related peptide (CGRP) exerts its diverse effects on vasodilation, nociception, secretion, and motor function through a heterodimeric receptor comprising of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). Despite the importance of CLR.RAMP1 in human disease, little is known about its distribution in the human gastrointestinal (GI) tract, where it participates in inflammation and pain. In this study, we determined that CLR and RAMP1 mRNAs are expressed in normal human stomach, ileum and colon by RT-PCR. We next characterized antibodies that we generated to rat CLR and RAMP1 in transfected HEK cells. Having characterized these antibodies in vitro, we then localized CLR-, RAMP1-, CGRP- and intermedin-immunoreactivity (IMD-IR) in various human GI segments. In the stomach, nerve bundles in the myenteric plexus and nerve fibers throughout the circular and longitudinal muscle had prominent CLR-IR. In the proximal colon and ileum, CLR was found in nerve varicosities of the myenteric plexus and surrounding submucosal neurons. Interestingly, CGRP expressing fibers did not co-localize, but were in close proximity to CLR. However, CLR and RAMP1, the two subunits of a functional CGRP receptor were clearly localized in myenteric plexus, where they may form functional cell-surface receptors. IMD, another member of calcitonin peptide family was also found in close proximity to CLR, and like CGRP, did not co-localize with either CLR or RAMP1 receptors. Thus, CGRP and IMD appear to be released locally, where they can mediate their effect on their receptors regulating diverse functions such as inflammation, pain and motility.

Endothelin-converting enzyme-1 regulates endosomal sorting of calcitonin receptor-like receptor and beta-arrestins

Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of endothelin-converting enzyme-1 (ECE-1a-d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), beta-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and beta-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and beta-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B(2) receptor, which transiently interacts with beta-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/beta-arrestin complex, freeing internalized receptors from beta-arrestins and promoting recycling and resensitization.

Post-endocytic sorting of calcitonin receptor-like receptor and receptor activity-modifying protein 1

Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene-related peptide (CGRP). Although CGRP induces endocytosis of CLR/RAMP1, little is known about post-endocytic sorting of these proteins. We observed that the duration of stimulation with CGRP markedly affected post-endocytic sorting of CLR/RAMP1. In HEK and SK-N-MC cells, transient stimulation (10(-7) M CGRP, 1 h), induced CLR/RAMP1 recycling with similar kinetics (2-6 h), demonstrated by labeling receptors in living cells with antibodies to extracellular epitopes. Recycling of CLR/RAMP1 correlated with resensitization of CGRP-induced increases in [Ca(2+)](i). Cycloheximide did not affect resensitization, but bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPases, abolished resensitization. Recycling CLR and RAMP1 were detected in endosomes containing Rab4a and Rab11a, and expression of GTPase-defective Rab4aS22N and Rab11aS25N inhibited resensitization. After sustained stimulation (10(-7) M CGRP, >2 h), CLR/RAMP1 trafficked to lysosomes. RAMP1 was degraded approximately 4-fold more rapidly than CLR (RAMP1, 45% degradation, 5 h; CLR, 54% degradation, 16 h), determined by Western blotting. Inhibitors of lysosomal, but not proteasomal, proteases prevented degradation. Sustained stimulation did not induce detectable mono- or polyubiquitination of CLR or RAMP1, determined by immunoprecipitation and Western blotting. Moreover, a RAMP1 mutant lacking the only intracellular lysine (RAMP1K142R) internalized and was degraded normally. Thus, after transient stimulation with CGRP, CLR and RAMP1 traffic from endosomes to the plasma membrane, which mediates resensitization. After sustained stimulation, CLR and RAMP1 traffic from endosomes to lysosomes by ubiquitin-independent mechanisms, where they are degraded at different rates.

Pseudomonas aeruginosa elastase disables proteinase-activated receptor 2 in respiratory epithelial cells

Pseudomonas aeruginosa, a major lung pathogen in cystic fibrosis (CF) patients, secretes an elastolytic metalloproteinase (EPa) contributing to bacterial pathogenicity. Proteinase-activated receptor 2 (PAR2), implicated in the pulmonary innate defense, is activated by the cleavage of its extracellular N-terminal domain, unmasking a new N-terminal sequence starting with SLIGKV, which binds intramolecularly and activates PAR2. We show that EPa cleaves the N-terminal domain of PAR2 from the cell surface without triggering receptor endocytosis as trypsin does. As evaluated by measurements of cytosolic calcium as well as prostaglandin E(2) and interleukin-8 production, this cleavage does not activate PAR2, but rather disarms the receptor for subsequent activation by trypsin, but not by the synthetic receptor-activating peptide, SLIGKV-NH(2). Proteolysis by EPa of synthetic peptides representing the N-terminal cleavage/activation sequences of either human or rat PAR2 indicates that cleavages resulting from EPa activity would not produce receptor-activating tethered ligands, but would disarm PAR2 in regard to any further activating proteolysis by activating proteinases. Our data indicate that a pathogen-derived proteinase like EPa can potentially silence the function of PAR2 in the respiratory tract, thereby altering the host innate defense mechanisms and respiratory functions, and thus contributing to pathogenesis in the setting of a disease like CF.

Isolation and characterization of an AINTEGUMENTA-like gene in different coconut (Cocos nucifera L.) varieties from Sri Lanka

Development of an efficient tissue culture protocol in coconut is hampered by numerous technical constraints. Thus a greater understanding of the fundamental aspects of embryogenesis is essential. The role of AINTEGUMENTA-like genes in embryogenesis has been elucidated not only in model plants but also in economically important crops. A coconut gene, CnANT, that encodes two APETALA2 (AP2) domains and a conserved linker region similar to those of the BABY BOOM transcription factor was cloned, characterized, and its tissue specific expression was examined. The full-length cDNA of 1,780 bp contains a 1,425-bp open reading frame that encodes a putative peptide of 474 amino acids. The genomic DNA sequence includes 2,317 bp and consists of nine exons interrupted by eight introns. The exon/intron organization of CnANT is similar to that of homologous genes in other plant species. Analysis of differential tissue expression by real-time polymerase chain reaction indicated that CnANT is expressed more highly in in vitro grown tissues than in other vegetative tissues. Sequence comparison of the genomic sequence of CnANT in different coconut varieties revealed one single nucleotide polymorphism and one indel in the first exon and first intron, respectively, which differentiate the Tall group of trees from Dwarfs. The indel sequence, which can be considered a simple sequence repeats marker, was successfully used to distinguish the Tall and Dwarf groups as well as to develop a marker system, which may be of value in the identification of parental varieties that are used in coconut breeding programs in Sri Lanka.

The effect of pH on the self-assembly of a collagen derived peptide amphiphile

Transitions in nanostructure driven by pH are observed for a self-assembling peptide amphiphile (PA) with a cationic pentapeptide headgroup. At pH 3, the PA forms flat tape-like structures, while at pH 4 the PA assembles into twisted right handed structures. These twisted structures transform again to flat tape-like structures at pH 7. In complete contrast, spherical micelles are observed at pH 2. These changes in response to pH may be relevant to biological and pharmaceutical applications of this PA in skincare.

Influence of elastase on alanine-rich peptide hydrogels

The self-assembly of the alanine-rich amphiphilic peptides Lys(Ala)6Lys (KA6K) and Lys(Ala)6Glu (KA6E)with homotelechelic or heterotelechelic charged termini respectively has been investigated in aqueous solution. These peptides contain hexa-alanine sequences designed to serve as substrates for the enzyme elastase. Electrostatic repulsion of the lysine termini in KA6K prevents self-assembly, whereas in contrast KA6E is observed, through electron microscopy, to form tape-like fibrils, which based on X-ray scattering contain layers of thickness equal to the molecular length. The alanine residues enable efficient packing of the side-chains in a beta-sheet structure, as revealed by circular dichroism, FTIR and X-ray diffraction experiments. In buffer, KA6E is able to form hydrogels at sufficiently high concentration. These were used as substrates for elastase, and enzyme-induced de-gelation was observed due to the disruption of the beta-sheet fibrillar network. We propose that hydrogels of the simple designed amphiphilic peptide KA6E may serve as model substrates for elastase and this could ultimately lead to applications in biomedicine and regenerative medicine.

Peptide growth factors signal differentially through protein kinase C to extracellular signal-regulated kinases in neonatal cardiomyocytes

The extracellular signal-regulated kinases 1/2 (ERK1/2) are activated in cardiomyocytes by Gq protein-coupled receptors and are associated with induction of hypertrophy. Here, we demonstrate that, in primary cardiomyocyte cultures, ERK1/2 were also significantly activated by platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or fibroblast growth factor (FGF), but insulin, insulin-like growth factor 1 (IGF-1) and nerve growth factor (NGF) had relatively minor effects. PDGF, EGF or FGF increased cardiomyocyte size via ERK1/2, whereas insulin, IGF-1 or NGF had no effect suggesting minimum thresholds/durations of ERK1/2 signaling are required for the morphological changes associated with hypertrophy. Peptide growth factors are widely accepted to activate phospholipase C gamma1 (PLCgamma1) and protein kinase C (PKC). In cardiomyocytes, only PDGF stimulated tyrosine phosphorylation of PLCgamma1 and nPKCdelta. Furthermore, activation of ERK1/2 by PDGF, but not EGF, required PKC activity. In contrast, EGF substantially increased Ras.GTP with rapid activation of c-Raf, whereas stimulation of Ras.GTP loading by PDGF was minimal and activation of c-Raf was delayed. Our data provide clear evidence for differential coupling of PDGF and EGF receptors to the ERK1/2 cascade, and indicate that a minimum threshold/duration of ERK1/2 signaling is required for the development of cardiomyocyte hypertrophy.

Supra-molecular assembly of a lumican-derived peptide amphiphile enhances its collagen-stimulating activity

C16-YEALRVANEVTLN, a peptide amphiphile (PA) incorporating a biologically active amino acid sequence found in lumican, has been examined for its influence upon collagen synthesis by human corneal fibroblasts in vitro, and the roles of supra-molecular assembly and activin receptor-like kinase ALK receptor signaling in this effect were assessed. Cell viability was monitored using the Alamar blue assay, and collagen synthesis was assessed using Sirius red. The role of ALK signaling was studied by receptor inhibition. Cultured human corneal fibroblasts synthesized significantly greater amounts of collagen in the presence of the PA over both 7-day and 21-day periods. The aggregation of the PA to form nanotapes resulted in a notable enhancement in this activity, with an approximately two-fold increase in collagen production per cell. This increase was reduced by the addition of an ALK inhibitor. The data presented reveal a stimulatory effect upon collagen synthesis by the primary cells of the corneal stroma, and demonstrate a direct influence of supra-molecular assembly of the PA upon the cellular response observed. The effects of PA upon fibroblasts were dependent upon ALK receptor function. These findings elucidate the role of self-assembled nanostructures in the biological activity of peptide amphiphiles, and support the potential use of a self-assembling lumican derived PA as a novel biomaterial, intended to promote collagen deposition for wound repair and tissue engineering purposes