Molecular Details of a Coupled Binding and Folding Reaction between the Amyloid Precursor Protein and a Folded Domain.

Intrinsically disordered regions in proteins often function as binding motifs in protein-protein interactions. The mechanistic aspects and molecular details of such coupled binding and folding reactions, which involve formation of multiple noncovalent bonds, have been broadly studied theoretically, but experimental data are scarce. Here, using a combination of protein semisynthesis to incorporate phosphorylated amino acids, backbone amide-to-ester modifications, side chain substitutions, and binding kinetics, we examined the interaction between the intrinsically disordered motif of amyloid precursor protein (APP) and the phosphotyrosine binding (PTB) domain of Mint2. We show that the interaction is regulated by a self-inhibitory segment of the PTB domain previously termed ARM. The helical ARM linker decreases the association rate constant 30-fold through a fast pre-equilibrium between an open and a closed state. Extensive side chain substitutions combined with kinetic experiments demonstrate that the rate-limiting transition state for the binding reaction is governed by native and non-native hydrophobic interactions and hydrogen bonds. Hydrophobic interactions were found to be particularly important during crossing of the transition state barrier. Furthermore, linear free energy relationships show that the overall coupled binding and folding reaction involves cooperative formation of interactions with roughly 30% native contacts formed at the transition state. Our data support an emerging picture of coupled binding and folding reactions following overall chemical principles similar to those of folding of globular protein domains but with greater malleability of ground and transition states.

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.

Investigating the role of GXXXG motifs in helical folding and self-association of plasticins, Gly/Leu-rich antimicrobial peptides.

Plasticins (PTC) are dermaseptin-related antimicrobial peptides characterized by a large number of leucine and glycine residues arranged in GXXXG motifs that are often described to promote helix association within biological membranes. We report the structure and interaction properties of two plasticins, PTC-B1 from Phyllomedusa bicolor and a cationic analog of PTC-DA1 from Pachymedusa dacnicolor, which exhibit membrane-lytic activities on a broad range of microorganisms. Despite a high number of glycine, CD and NMR spectroscopy show that the two plasticins adopt mainly alpha-helical conformations in a wide variety of environments such as trifluoroethanol, detergent micelles and lipid vesicles. In DPC and SDS, plasticins adopt well-defined helices that lie parallel to the micelle surface, all glycine residues being located on the solvent-exposed face. Spectroscopic data and cross-linking experiments indicate that the GXXXG repeats in these amphipathic helices do not provide a strong oligomerization interface, suggesting a different role from GXXXG motifs found in transmembrane helices.

Systemic delivery of P42 peptide: a new weapon to fight Huntington's disease.

BACKGROUND: In Huntington's disease (HD), the ratio between normal and mutant Huntingtin (polyQ-hHtt) is crucial in the onset and progression of the disease. As a result, addition of normal Htt was shown to improve polyQ-hHtt-induced defects. Therefore, we recently identified, within human Htt, a 23aa peptide (P42) that prevents aggregation and polyQ-hHtt-induced phenotypes in HD Drosophila model. In this report, we evaluated the therapeutic potential of P42 in a mammalian model of the disease, R6/2 mice. RESULTS: To this end, we developed an original strategy for P42 delivery, combining the properties of the cell penetrating peptide TAT from HIV with a nanostructure-based drug delivery system (Aonys® technology), to form a water-in-oil microemulsion (referred to as NP42T) allowing non-invasive per mucosal buccal/rectal administration of P42. Using MALDI Imaging Mass Spectrometry, we verified the correct targeting of NP42T into the brain, after per mucosal administration. We then evaluated the effects of NP42T in R6/2 mice. We found that P42 (and/or derivatives) are delivered into the brain and target most of the cells, including the neurons of the striatum. Buccal/rectal daily administrations of NP42T microemulsion allowed a clear improvement of behavioural HD-associated defects (foot-clasping, rotarod and body weights), and of several histological markers (aggregation, astrogliosis or ventricular areas) recorded on brain sections. CONCLUSIONS: These data demonstrate that NP42T presents an unprecedented protective effect, and highlight a new therapeutic strategy for HD, associating an efficient peptide with a powerful delivery technology.

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.

One-pot semisynthesis of exon 1 of the Huntingtin protein: new tools for elucidating the role of posttranslational modifications in the pathogenesis of Huntington's disease.

The natural enzymes involved in regulating many of the posttranslational modifications (PTMs) within the first 17 residues (Nt17) of Huntingtin exon 1 (Httex1) remain unknown. A semisynthetic strategy that allows the site-specific introduction of PTMs within Nt17 by using expressed protein ligation (EPL) was developed. This strategy was used to produce untagged wild-type (wt) and T3-phosphorylated (pT3) Httex1 containing 23 glutamine residues (Httex1-23Q). Our studies show that pT3 significantly slows the oligomerization and fibrillization of Httex1-23Q and that Httex1 variants containing polyQ repeats below the pathogenic threshold readily aggregate and form fibrils in vitro. These findings suggest that crossing the polyQ pathogenic threshold is not essential for Httex1 aggregation. The ability to produce wt or site-specifically modified tag-free Httex1 should facilitate determining its structure and the role of N-terminal PTMs in regulating the functions of Htt in health and disease.

Chemical synthesis and orexigenic activity of rat/mouse relaxin-3.

The insulin-like peptide, relaxin-3 was first identified just a decade ago via a genomic database search and is now recognized to be a key neuropeptide with several roles including the regulation of arousal, stress responses and neuroendocrine homeostasis. It also has significant potential as a drug to treat stress and obesity. Its actions are mediated via its cognate G protein-coupled receptor, RXFP3, which is found in abundant numbers in the brain. However, much remains to be determined with respect to the mechanism of neurological action of this peptide. Consequently, the chemical synthesis of the rat and mouse (which share identical primary structures) two-chain, three disulfide peptide was undertaken and the resulting peptide subjected to detailed in vitro and in vivo assay. Use of efficient solid-phase synthesis methods provided the two regioselectively S-protected A- and B-chains which were readily combined via sequential disulfide bond formation. The synthetic rat/mouse relaxin-3 was obtained in high purity and good overall yield. It demonstrated potent orexigenic activity in rats in that central intracerebroventricular infusion led to significantly increased food intake and water drinking.

Synthesis of the IGF-II-like hormone vesiculin using regioselective formation of disulfide bonds.

Diabetes mellitus, characterised by hyperglycemia and altered ß-cell function, is an increasingly common disorder affecting millions of individuals world-wide. While therapeutic regimens exist to manage the condition, diabetic individuals remain prone to complications that are detrimental to both their length and quality of life. An improved understanding of the disease which may then enable development of new treatments is therefore a desirable goal. Vesiculin, a novel IGF-II-like protein was recently isolated from the secretory granules of murine ß-cells, and preliminary studies indicate it is capable of signalling via the insulin receptor (IR)/insulin-like growth factor receptor 1(IGF1R) family giving it the potential to elicit both metabolic and mitogenic responses in the beta-cell. In order to facilitate further studies on this new member of the insulin-family of hormones, we undertook a chemical synthesis of the protein using regioselective disulfide bond formation.

Natural and synthetic chiral isoforms of crustacean hyperglycemic hormone from the crayfish Astacus leptodactylus: hyperglycemic activity and hemolymphatic clearance.

In the crayfish Astacus leptodactylus, as in several crustacean species, the crustacean hyperglycemic hormone is present as two isoforms differing by the chirality of the third residue, a phenylalanine. In the present work, isoforms synthesized full length by solid-phase peptide synthesis have been purified, refolded, the location of the disulfide bridges has been checked, their immunoreactivity against different antibodies have been analyzed and their hyperglycemic activity tested, to ensure the identity of the synthetic peptides with their natural homologs. Different parameters of the hyperglycemic activity of both isoforms were studied. In addition to a difference in the kinetics of hyperglycemia, already known from other studies, it was observed that the dose-response was different depending on the season where experiments were performed, the response being stronger in spring than in autumn, especially for the d-Phe containing isoform. A dosage method based on sandwich enzyme linked immunosorbent assay (ELISA) has been developed to measure hemolymphatic levels of the isoforms after spiking of the animals with one isoform or the other. It was found that hemolymphatic clearance was identical for both isoforms, indicating that their differential effect is not linked to their different lifetime in the hemolymph but may rather rely on other mechanisms such as their binding to different target tissues.

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.

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.

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.

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.

Syntheses and binding affinities of 6-nitroquipazine analogues for serotonin transporter: Part 3. A potential 5-HT transporter imaging agent, 3-(3-[18F]fluoropropyl)-6-nitroquipazine.

3-(3-[18F]Fluoropropyl)-6-nitroquipazine ([18F]FPNQ) as a 5-HT transporter imaging agents was designed, synthesized, and evaluated. FPNQ was selected due to its potent in vitro biological activity (K(i)=0.32 nM) in rat brain cortical membranes. The 18F-labeled FPNQ was prepared by reaction of the propyl mesylate as a precursor with tetra-n-butylammonium [18F]fluoride generated under NCA conditions. The precursor mesylate was synthesized from commercially available hydrocarbostyril in nine steps in 21% overall yield. The specific activity of the [18F]FPNQ determined by radioreceptor assay was 27.0 GBq/micromol. Tissue distribution studies in mice showed the highest uptake in the frontal cortex (5.79 %ID/g) at 60 min post-injection.

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.