Antimicrobial peptides Pep19-2.5 and Pep19-4LF inhibit Streptococcus mutans growth and biofilm formation.

With this study, we investigated the effect of synthetic antimicrobial peptides Pep19-2.5 and Pep194LF alone or in combination with antibiotics on S. mutans growth and biofilm formation/disruption. We also examined the cytotoxic effect of each peptide on monocytes. S. mutans was cultured in the presence of different concentrations of each peptide. We showed that Pep19-2.5 and Pep19-4LF were able to significantly (p ≤ 0.01) inhibit the growth of S. mutans. The synthetic peptides also decreased biofilm formation by S. mutans. Furthermore, both peptides reduced the viability of S. mutans in already formed biofilms. The combination of each peptide with antibiotics (penicillin/streptomycin, P/S) produced additive interactions which inhibited S. mutans growth and biofilm formation. Pep19-2.5 and Pep19-4LF were nontoxic, as they did not decrease monocyte viability and did not increase the lactate dehydrogenase activity of the exposed cells. In conclusion, synthetic peptides Pep19-2.5 and Pep19-4LF did inhibit S. mutans growth and its capacity to form biofilm. Both peptides were found to be nontoxic to monocytes. These data provide new insight into the efficacy of synthetic peptides Pep19-2.5 and Pep19-4LF against S. mutans. These peptides may thus be useful in controlling the adverse effects of this cariogenic bacterium in human.

Microwave-assisted solid-phase peptide synthesis of neurosecretory protein GL composed of 80 amino acid residues.

We recently identified a novel cDNA encoding a small secretory protein of 80 amino acid residues, termed neurosecretory protein GL (NPGL), from the chicken hypothalamus. Homologs of NPGL have been reported to be present in mammals, such as human and rat. NPGL is amidated at its C-terminus, contains an intramolecular disulfide bond, and is hydrophobic in nature. In this study, we have optimized the synthesis of the entire 80-amino acid peptide sequence of rat NPGL by microwave-assisted solid-phase peptide synthesis. NPGL was obtained with a 10% yield when the coupling reactions were performed using 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate (HATU) at 50 °C for 5 min, and Fmoc deprotections were performed using 40% piperidine containing 0.1 M HOBt. Furthermore, the disulfide bond of NPGL was formed with 20% yield with the use of glutathione-containing redox buffer and 50% acetonitrile.

Aggregation behavior of chemically synthesized, full-length huntingtin exon1.

Repeat length disease thresholds vary among the 10 expanded polyglutamine (polyQ) repeat diseases, from about 20 to about 50 glutamine residues. The unique amino acid sequences flanking the polyQ segment are thought to contribute to these repeat length thresholds. The specific portions of the flanking sequences that modulate polyQ properties are not always clear, however. This ambiguity may be important in Huntington's disease (HD), for example, where in vitro studies of aggregation mechanisms have led to distinctly different mechanistic models. Most in vitro studies of the aggregation of the huntingtin (HTT) exon1 fragment implicated in the HD mechanism have been conducted on inexact molecules that are imprecise either on the N-terminus (recombinantly produced peptides) or on the C-terminus (chemically synthesized peptides). In this paper, we investigate the aggregation properties of chemically synthesized HTT exon1 peptides that are full-length and complete, containing both normal and expanded polyQ repeat lengths, and compare the results directly to previously investigated molecules containing truncated C-termini. The results on the full-length peptides are consistent with a two-step aggregation mechanism originally developed based on studies of the C-terminally truncated analogues. Thus, we observe relatively rapid formation of spherical oligomers containing from 100 to 600 HTT exon1 molecules and intermediate formation of short protofibril-like structures containing from 500 to 2600 molecules. In contrast to this relatively rapid assembly, mature HTT exon1 amyloid requires about one month to dissociate in vitro, which is similar to the time required for neuronal HTT exon1 aggregates to disappear in vivo after HTT production is discontinued.

Novel protocol for the chemical synthesis of crustacean hyperglycemic hormone analogues--an efficient experimental tool for studying their functions.

The crustacean Hyperglycemic Hormone (cHH) is present in many decapods in different isoforms, whose specific biological functions are still poorly understood. Here we report on the first chemical synthesis of three distinct isoforms of the cHH of Astacus leptodactylus carried out by solid phase peptide synthesis coupled to native chemical ligation. The synthetic 72 amino acid long peptide amides, containing L- or D-Phe³ and (Glp¹, D-Phe³) were tested for their biological activity by means of homologous in vivo bioassays. The hyperglycemic activity of the D-isoforms was significantly higher than that of the L-isoform, while the presence of the N-terminal Glp residue had no influence on the peptide activity. The results show that the presence of D-Phe³ modifies the cHH functionality, contributing to the diversification of the hormone pool.

Inhibiting the nucleation of amyloid structure in a huntingtin fragment by targeting α-helix-rich oligomeric intermediates.

Although oligomeric intermediates are transiently formed in almost all known amyloid assembly reactions, their mechanistic roles are poorly understood. Recently, we demonstrated a critical role for the 17-amino-acid N-terminus (htt(NT) segment) of huntingtin (htt) in the oligomer-mediated amyloid assembly of htt N-terminal fragments. In this mechanism, the htt(NT) segment forms the α-helix-rich core of the oligomers, leaving much of the polyglutamine (polyQ) segment disordered and solvent-exposed. Nucleation of amyloid structure occurs within this local high concentration of disordered polyQ. Here we demonstrate the kinetic importance of htt(NT) self-assembly by describing inhibitory htt(NT)-containing peptides that appear to work by targeting nucleation within the oligomer fraction. These molecules inhibit amyloid nucleation by forming mixed oligomers with the htt(NT) domains of polyQ-containing htt N-terminal fragments. In one class of inhibitors, nucleation is passively suppressed due to the reduced local concentration of polyQ within the mixed oligomer. In the other class, nucleation is actively suppressed by a proline-rich polyQ segment covalently attached to htt(NT). Studies with D-amino acid and scrambled sequence versions of htt(NT) suggest that inhibition activity is strongly linked to the propensity of inhibitory peptides to make amphipathic α-helices. Htt(NT) derivatives with C-terminal cell-penetrating peptide segments also exhibit excellent inhibitory activity. The htt(NT)-based peptides described here, especially those with protease-resistant d-amino acids and/or with cell-penetrating sequences, may prove useful as lead therapeutics for inhibiting the nucleation of amyloid formation in Huntington's disease.

Synthesis of N-terminally linked protein and peptide dimers by native chemical ligation.

Dimerization can be utilized to double the molecular weight of proteins and peptides and potentially increase their avidity of binding to target receptors. These dimerization effects may be utilized to increase in vivo half-lives in a manner similar to PEGylation and may also improve biological activity. In this paper, we report a new strategy for the synthesis of N-terminally linked protein and peptide homodimers utilizing native chemical ligation to conjugate a short dithioester linker to the N-terminal cysteines of protein and peptide monomers to form dimers in a single step. This strategy is general and has been applied to the production of dimers from three recombinantly expressed polypeptides, the IgG binding domain Protein G, an HIV entry inhibitor peptide C37H6, and human interleukin-1 receptor antagonist (IL-1ra). The biological activities of the C37H6 and IL-1ra dimers produced by these methods were retained or even slightly increased when compared to their corresponding monomers.

Synthesis of pathological and nonpathological human exon 1 huntingtin.

Huntington's disease (HD) is a neurodegenerative disorder that affects approximately 1 in 10 000 individuals. The underlying gene mutation was identified as a CAG-triplet repeat expansion in the gene huntingtin. The CAG sequence codes for glutamine, and in HD, an expansion of the polyglutamine (poly-Q) stretch above 35 glutamine residues results in pathogenicity. It has been demonstrated in various animal models that only the expression of exon 1 huntingtin, a 67-amino acid-long polypeptide plus a variable poly-Q stretch, is sufficient to cause full HD-like pathology. Therefore, a deeper understanding of exon 1 huntingtin, its structure, aggregation mechanism and interaction with other proteins is crucial for a better understanding of the disease. Here, we describe the synthesis of a 109-amino acid-long exon 1 huntingtin peptide including a poly-Q stretch of 42 glutamines. This microwave-assisted solid phase peptide synthesis resulted in milligram amounts of peptide with high purity. We also synthesized a nonpathogenic version of exon 1 huntingtin (90-amino acid long including a poly-Q stretch of 23 glutamine residues) using the same strategy. In circular dichroism spectroscopy, both polypeptides showed weak alpha-helical properties with the longer peptide showing a higher helical degree. These model peptides have great potential for further biomedical analyses, e.g. for large-scale pre-screenings for aggregation inhibitors, further structural analyses as well as protein-protein interaction studies.

Differential effects of Phe19 and Phe20 on fibril formation by amyloidogenic peptide A beta 16-22 (Ac-KLVFFAE-NH2).

The sequence KLVFFAE (A beta 16-22) in Alzheimer's beta-amyloid is thought to be a core beta-structure that could act as a template for folding other parts of the polypeptide or molecules into fibrillar assemblies rich in beta-sheet. To elucidate the mechanism of the initial folding process, we undertook combined X-ray fiber/powder diffraction and infrared (IR) spectroscopy to analyze lyophilized A beta 16-22 and solubilized/dried peptide containing nitrile probes at F19 and/or F20. Solubilized/dried wild-type (WT) A beta 16-22 and the peptide containing cyanophenylalanine at F19 (19CN) or at F20 (20CN) gave fiber patterns consistent with slab-like beta-crystallites that were cylindrically averaged around the axis parallel to the polypeptide chain direction. The WT and 19CN assemblies showed 30-A period arrays arising from the stacking of the slabs along the peptide chain direction, whereas the 20CN assemblies lacked any such stacking. The electron density projection along the peptide chain direction indicated similar side-chain dispositions for WT and 20CN, but not for 19CN. These X-ray results and modeling imply that in the assembly of WT A beta 16-22 the F19 side chain is localized within the intersheet space and is involved in hydrophobic contact with amino acids across the intersheet space, whereas the F20 side chain localized near the slab surface is less important for the intersheet interaction, but involved in slab stacking. IR observations for the same peptides in dilute solution showed a greater degree of hydrogen bonding for the nitrile groups in 20CN than in 19CN, supporting this interpretation.

Tandem ligation at X-Cys and Gly-Gly positions via an orthogonally protected auxiliary group.

4,5-dimethoxy-2-mercaptobenzylamine (Dmmb) has been protected by acetamidomethyl (Acm) and incorporated into a peptide thioester for use in tandem native chemical ligation. Upon ligation between the thioester and a Cys-peptide, Acm was removed from Dmmb using silver acetate, and a second ligation reaction was done at the Dmmb position. Dmmb removal using TFMSA-TFA effected overall tandem ligation at X-Cys and Gly-Gly.

Solid-phase synthesis and purification of a set of uniformly 13C, 15N labelled de novo designed membrane fusogenic peptides.

The transmembrane segments of soluble N-ethylmaleimide-sensitive factor (SNARE) proteins or viral envelope proteins drive membrane fusion, which suggests that simple synthetic biology constructs for fusion exist and can be evaluated. We describe the high-yield synthesis of a set of de novo designed fusogenic peptides for use in functional investigations, which are highly enriched in 13C and 15N using three equivalents of labelled amino acids and optimized reaction conditions minimizing aggregation. The biomimetic peptides have a high purity >90% and show reproducible and fusogenic activity that correlates well with the intended functional design characteristics, from strongly fusogenic to almost non-fusogenic.

Teneurin proteins possess a carboxy terminal sequence with neuromodulatory activity.

We have previously shown that a bioactive neuropeptide-like sequence is present at the carboxy-terminus of the teneurin transmembrane proteins. We have subsequently called this peptide 'teneurin C-terminal associated peptide' (TCAP). The sequence encodes a peptide 40 or 41 amino acids long flanked by a cleavage motif on the amino terminus and an amidation motif on the carboxy terminus, characteristic of bioactive peptides. This sequence is highly conserved in all vertebrates. A TCAP-like sequence is encoded by each of the four teneurin genes. We have therefore examined the neurological role TCAP-1 may play in mice and rats. In situ hybridization studies showed that the teneurin-1 mRNA containing the TCAP-1 sequence is expressed in regions of the forebrain and limbic system regulating stress and anxiety. A synthetic version of amidated mouse/rat TCAP-1 was prepared by solid-phase synthesis and used to investigate the in vitro and in vivo activity. TCAP-1 induces a dose-dependent change in cAMP accumulation and MTT activity in immortalized mouse neurons. Administration of synthetic TCAP-1 into the basolateral amygdala significantly increases the acoustic startle response in low-anxiety rats and decreases the response in high-anxiety animals in a dose-dependent manner. When 30 pmol TCAP-1 is administered into the lateral ventricles each day for 5 days, the sensitization of the rats to the acoustic startle response is abolished. These data indicate that TCAP may possess functions that are independent of the teneurin proprotein and together, the teneurins and TCAP, may represent a novel system to regulate neuronal function and emotionality.

Development of an antibody against a 40,000 mol. wt brain injury-derived neurotrophic peptide-binding protein and identification of a 40,000 mol. wt brain injury-derived neurotrophic peptide-binding protein in hippocampal neurons.

Brain injury-derived neurotrophic peptide is a 13-amino acid peptide derived from a 15,000 mol. wt neurotrophic factor released from sites of mechanical injury in neonatal rat brain. This peptide promotes survival of septal cholinergic neurons and mesencephalic dopaminergic neurons, and protects hippocampal neurons from glutamate-induced neurotoxicity. In this study, we have developed a monoclonal antibody against a brain injury-derived neurotrophic peptide-binding protein by immunizing mice with septal synaptosomes from five-week-old rat brain. Monoclonal antibodies were screened for inhibition of the binding of a 125I-labeled analogue of brain injury-derived neurotrophic peptide to rat brain synaptosomes. The monoclonal antibody 6A22 suppressed the biological activity of brain injury-derived neurotrophic peptide and abolished the protective effect of the neurotrophic peptide against glutamate-induced neurotoxicity. This monoclonal antibody recognized a 40,000 mol. wt brain injury-derived neurotrophic peptide-binding protein, which was also identified by cross-linking experiments. Immunohistochemical studies showed that the 6A22 antibody bound to the cell surfaces of a subpopulation (about 60%) of hippocampal neurons in culture. These results are consistent with the possibility that the 40,000 mol. wt protein belongs to brain injury-derived neurotrophic peptide receptors.

Síntesis y liberación in vivo de proteínas en el sistema nervioso central de la rata / In vivo release of newly synthetized protein-like material from the striatum in the freely moving rat

Previamente informamos sobre la liberación inducida por despolarización del aminoácido inhibitorio GABA y del péptido modulador encefalina (bayón y col. 1986). Un hallazgo interesante observado durante el curso de estos experimentos es el hecho de que sustancias de alto peso molecular cuantificadas por el método de Lowry, son liberadas in vivo en condiciones basales y por estimulación despolarizante del tejido nervioso de la rata con K+ 50 mM. Dentro de este contexto, Bayón (1986) observó que la liberación inducida del material protéico con K+ 50 mM en el estriado de la rata, era parcialmente dependiente de la concentración de Ca++ en el líquido de perfusión (1.8 mM), lo que sugería que la secreción neuronal de este material protéico de alto peso molecular, se libera por los mecanismos fisiológicos establecidos de acoplamiento "estimulo-secreción" que se dan para los transmisores peptídicos y para los transmisores no peptídicos. Sin embargo, el origen de este material protéico, liberado bajo estimulación química despolarizante del tejido nervioso, aún queda por elucidarse, puesto que este material pudiera tener un origen plasmático mediante la extravasación de proteínas del compartimiento vascular al espacio interneuronal. Dentro de este contexto exploramos la hipótesis de que este material protéico pudiera tener un origen neuronal exclusivo, mediante la aplicación local de un precursor metabólico, como es el aminoácido S-Metionina al estradio de la rata. Nuestros resultado muestran que sólo una pequeña fracción del material protéico sintetizado in situ y precipitado por TCA, es liberado en condiciones basales y en condiciones de despolarización química del tejido nervioso durante la perfusión in vivo del estriado de la rata. Estos resultados preliminares sugieren, por lo menos, que este material de alto peso molecular liberado al espacio sináptico pudiera ejercer funciones importantes en la comunicación química interneuronal, tal como se ha encontrado en otras especies de alto peso molecular, como la AchE y la dopamina-beta-hidroxilasa(DBH)

Solid-phase synthesis, conformational analysis and in vitro cleavage of synthetic human synaptobrevin II 1-93 by tetanus toxin L chain.

A 93-residue peptide corresponding to the cytosolic domain of a human vesicle associated membrane protein (VAMP or synaptobrevin) has been prepared by solid-phase peptide synthesis in order to investigate the proteolytic activity of the tetanus toxin light chain (TeTx L chain). This protein has been recently reported to inactivate the neuronal rat synaptobrevin II by proteolysis. We show in this study that the synthetic human synaptobrevin II 1-93 (Syb II 1-93) as well as an N-terminus-shortened 69-residue peptide (Syb II 25-93) were cleaved selectively at the Gln76-Phe77 peptide bond by TeTx L chain while shorter peptides were not. A Michaelis constant Km = 192 +/- 2 microM and a catalytic constant kcat = 0.5 min-1 were found for the 93-residue peptide. A neutral optimum pH for the cleavage rate, an inhibition by preincubation of the toxin with well known nonspecific inhibitors of metallopeptidases as well as a zinc-dependent enzyme activity suggest that TeTx belongs to the zinc endopeptidase family. Moreover an activation by reducing agents and an inhibition by cysteine-modifying chemical reagents indicate a critical thiol dependency. Among several specific inhibitors of zinc endopeptidases tested, none could inhibit TeTx L chain even at high concentration. Structural studies by 600-MHz 1H-NMR showed that in water or dimethylsulfoxide the peptide Syb II 1-93 and shorter fragments did not present well defined conformations. Nevertheless protein-protein interactions have been shown for the peptides Syb II 1-93 and 25-93 but not for Syb II 51-93, a fragment not cleaved by TeTx L chain.

Stimulation of insulin release from permeabilized HIT-T15 cells by a synthetic peptide corresponding to the effector domain of the small GTP-binding protein rab3.

A synthetic peptide (rab3AL) corresponding to the effector domain of rab3, a small GTP-binding protein, stimulated basal and potentiated Ca(2+)- as well as GTP gamma S-evoked insulin secretion about 2-fold from streptolysin-O permeabilized HIT cells. This effect was specific, since the analogous peptides of ras or rab1 did not affect the exocytotic event. The more than additive effect of rab3AL on Ca2+ or GTP gamma S stimulation indicates a distinct mode of action of the peptide. The partial loss of cytosolic proteins from permeabilized cells was accompanied by a faster run-down of the secretory response to Ca2+ than the one to GTP gamma S. The persistent effect of rab3AL under these conditions points to a membrane localization of its target. These results suggest that rab3 and its effector are involved in the regulation of insulin secretion.

Efficient solid phase peptide synthesis. Use of methanesulfonic acid alpha-amino deprotecting procedure and new coupling reagent, 2-(benzotriazol-1-yl)oxy-1,3-dimethylimidazolidinium hexafluorophosphate (BOI).

An efficient method for solid phase peptide synthesis was developed, which consists of N alpha-selective deprotection by dilute methanesulfonic acid, in situ neutralization and rapid coupling reaction using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or 2-(benzotriazol-1-yl)oxy-1,3- dimethylimidazolidinium hexafluorophosphate (BOI) reagent. Selective removal of the N alpha-Boc group by dilute methanesulfonic acid was of more advantage than removal by TFA in terms of stability of semipermanent protecting groups and suppression of undesired side reactions. The use of in situ neutralization and rapid coupling method reduced intramolecular aminolytic cyclization by shortening exposure of the deprotected nucleophilic amino group. A successful synthesis of porcine brain natriuretic peptide (pBNP) has been achieved using this efficient solid phase peptide synthesis scheme.

Determination of disulfide bond arrangement in bombyxin-IV, an insulin superfamily peptide from the silkworm, Bombyx mori, by combination of thermolysin digestion of natural peptide and selective synthesis of disulfide bond isomers.

The mode of disulfide linkages in bombyxin-IV, an insulin superfamily peptide consisting of A- and B-chains, was determined as A6-A11, A7-B10, and A20-B22. An intermolecular bond of A20-B22 was identified by sequencing and mass spectrometric analysis of the fragments generated by thermolysin digestion of natural bombyxin-IV. The mode of the remaining two bridges was determined by chemical and selective synthesis of three possible disulfide bond isomers of bombyxin-IV. A- and B-chains were synthesized by solid-phase method, and three disulfide bonds were bridged stepwise and in a fully controlled manner. Retention time on reversed-phase high-performance liquid chromatography (HPLC), thermolysin digests, and biological activity of the synthetic [A6-A11, A7-B10, A20-B22-cystine]-bombyxin-IV revealed that it was identical with the natural bombyxin-IV. Two other isomers with respect to disulfide bond arrangement, [A6-A7, A11-B10, A20-B22-cystine]- and [A6-B10, A7-A11, A20-B22-cystine]-bombyxin-IVs, were distinguishable from the natural one by use of HPLC, thermolysin digestion, and bioassay.

Solution-phase synthesis of porcine brain natriuretic peptide (pBNP) using S-trimethylacetamidomethylcysteine.

The hexadodecapeptide corresponding to the entire amino acid sequence of porcine brain natriuretic peptide (pBNP) was synthesized by assembling four segments in solution, followed by HF deprotection and subsequent oxidation to establish an intramolecular disulfide bridge. The synthesis using the newly developed S-trimethylacetamidomethylcysteine [Cys(Tacm)] derivative gave a better yield than that using the S-2,4,6-trimethylbenzylcysteine [Cys(Tmb)] derivative. The chick rectum relaxant activity of the synthetic pBNP was 2.9 times more potent than that of alpha-rat atrial natriuretic peptide (alpha-rANP).

Characterization of immunoreactive brain natriuretic peptide in human cardiac atrium.

Based on cDNA sequence data for human brain natriuretic peptide (BNP) precursor (1), a radioimmunoassay (RIA) system highly specific to human BNP (hBNP) was developed and used to characterize immunoreactive (ir-) hBNP in cardiac atrium. Gel filtration of ir-hBNP in atrium indicated that ir-hBNP was mainly comprised of two molecular forms of 13-15K and 4K. In reverse phase high performance liquid chromatography (HPLC), the low molecular weight (MW) ir-hBNP emerged as a single peak at an elution time identical to that of synthetic hBNP-32. The high MW ir-hBNP was also eluted as a single peak. On the other hand, tissue concentrations of ir-hBNP in cardiac atria were found to be 9.98-593.22 pmol/g in 13 specimens, being about 1/150 the concentration of ir-human atrial natriuretic peptide (hANP). These results demonstrate that hBNP is present as a peptide in human heart, suggesting that hBNP is secreted from heart and functions together with hANP as a hormone.

Synthesis of bombyxin-IV, an insulin-like heterodimeric peptide from the silkworm, Bombyx mori.

Bombyxin-IV, a molecular species of bombyxin, which is a member of insulin-like heterodimeric peptides of the silkworm Bombyx mori with prothoracicotropic hormone activity, was synthesized. The A- and B-chains of bombyxin-IV containing four and two Cys residues, respectively, were first synthesized separately by solid phase chemistry using Boc protocol. Then they were coupled by stepwise removal of two different protecting groups at the cysteinyl thiols for semiselective formation of disulfide bridges to give bombyxin-IV in 8% yield. The synthetic bombyxin-IV was shown to have chromatographic and biological properties identical with those of natural bombyxin-IV.