Tachykinin Neuropeptides and Amyloid ß (25-35) Assembly: Friend or Foe?

Amyloid ß (Aß) protein is responsible for Alzheimer's disease, and one of its important fragments, Aß(25-35), is found in the brain and has been shown to be neurotoxic. Tachykinin neuropeptides, including Neuromedin K (NK), Kassinin, and Substance P, have been reported to reduce Aß(25-35)'s toxicity in cells even though they share similar primary structures with Aß(25-35). Here, we seek to understand the molecular mechanisms of how these peptides interact with Aß(25-35) and to shed light on why some peptides with similar primary structures are toxic and others nontoxic. We use both experimental and computational methods, including ion mobility mass spectrometry and enhanced-sampling replica-exchange molecular dynamics simulations, to study the aggregation pathways of Aß(25-35), NK, Kassinin, Substance P, and mixtures of the latter three with Aß(25-35). NK and Substance P were observed to remove the higher-order oligomers (i.e., hexamers and dodecamers) of Aß(25-35), which are related to its toxicity, although Substance P did so more slowly. In contrast, Kassinin was found to promote the formation of these higher-order oligomers. This result conflicts with what is expected and is elaborated on in the text. We also observe that even though they have significant structural homology with Aß(25-35), NK, Kassinin, and Substance P do not form hexamers with a ß-sheet structure like Aß(25-35). The hexamer structure of Aß(25-35) has been identified as a cylindrin, and this structure has been strongly correlated to toxic species. The reasons why the three tachykinin peptides behave so differently when mixed with Aß(25-35) are discussed.

Oligomer Formation of Toxic and Functional Amyloid Peptides Studied with Atomistic Simulations.

Amyloids are associated with diseases, including Alzheimer's, as well as functional roles such as storage of peptide hormones. It is still unclear what differences exist between aberrant and functional amyloids. However, it is known that soluble oligomers formed during amyloid aggregation are more toxic than the final fibrils. Here, we perform molecular dynamics simulations to study the aggregation of the amyloid-ß peptide Aß25-35, associated with Alzheimer's disease, and two functional amyloid-forming tachykinin peptides: kassinin and neuromedin K. Although the three peptides have similar primary sequences, tachykinin peptides, in contrast to Aß25-35, form nontoxic amyloids. Our simulations reveal that the charge of the C-terminus is essential to controlling the aggregation process. In particular, when the kassinin C-terminus is not amidated, the aggregation kinetics decreases considerably. In addition, we observe that the monomeric peptides in extended conformations aggregate faster than those in collapsed hairpin-like conformations.

Amyloid-like fibril formation by tachykinin neuropeptides and its relevance to amyloid ß-protein aggregation and toxicity.

Protein aggregation and amyloid formation are associated with both pathological conditions in humans such as Alzheimer's disease and native functions such as peptide hormone storage in the pituitary secretory granules in mammals. Here, we studied amyloid fibrils formation by three neuropeptides namely physalaemin, kassinin and substance P of tachykinin family using biophysical techniques including circular dichroism, thioflavin T, congo red binding and microscopy. All these neuropeptides under study have significant sequence similarity with Aß(25-35) that is known to form neurotoxic amyloids. We found that all these peptides formed amyloid-like fibrils in vitro in the presence of heparin, and these amyloids were found to be nontoxic in neuronal cells. However, the extent of amyloid formation, structural transition, and morphology were different depending on the primary sequences of peptide. When Aß(25-35) and Aß40 were incubated with each of these neuropeptides in 1:1 ratio, a drastic increase in amyloid growths were observed compared to that of individual peptides suggesting that co-aggregation of Aß and these neuropeptides. The electron micrographs of these co-aggregates were dissimilar when compared with individual peptide fibrils further supporting the possible incorporation of these neuropeptides in Aß amyloid fibrils. Further, the fibrils of these neuropeptides can seed the fibrils formation of Aß40 and reduced the toxicity of preformed Aß fibrils. The present study of amyloid formation by tachykinin neuropeptides is not only providing an understanding of the mechanism of amyloid fibril formation in general, but also offering plausible explanation that why these neuropeptide might reduce the cytotoxicity associated with Alzheimer's disease related amyloids.

Kassina senegalensis skin tachykinins: molecular cloning of kassinin and (Thr2, Ile9)-kassinin biosynthetic precursor cDNAs and comparative bioactivity of mature tachykinins on the smooth muscle of rat urinary bladder.

Tachykinins are among the most widely-studied families of regulatory peptides characterized by a highly-conserved C-terminal -Phe-X-Gly-Leu-Met.amide motif, which also constitutes the essential bioactive core. The amphibian skin has proved to be a rich source of these peptides with physalaemin from the skin of Physalaemus fuscomaculatus representing the archetypal aromatic tachykinin (X=Tyr or Phe) and kassinin from the skin of Kassina senegalensis representing the archetypal aliphatic tachykinin in which X=Val or Ile. Despite the primary structures of both mature peptides having been known for at least 30 years, neither the structures nor organizations of their biosynthetic precursors have been reported. Here we report the structure and organization of the biosynthetic precursor of kassinin deduced from cDNA cloned from a skin secretion library. In addition, a second precursor cDNA encoding the novel kassinin analog (Thr(2), Ile(9))-kassinin was identified as was the predicted mature peptide in skin secretion. Both transcripts exhibited a high degree of nucleotide sequence similarity and of open-reading frame translated amino acid sequences of putative precursor proteins. The translated preprotachykinins each consisted of 80 amino acid residues encoding single copies of either kassinin or its site-substituted analog. Synthetic replicates of each kassinin were found to be active on rat urinary bladder smooth muscle at nanomolar concentrations. The structural organization of both preprotachykinins differs from that previously reported for those of Odorrana grahami skin indicating a spectrum of diversity akin to that established for amphibian skin preprobradykinins.

Lipid induced conformation of the tachykinin peptide Kassinin.

Both the aqueous and lipid-induced structure of Kassinin, a dodecapeptide of amphibian origin, has been studied by two-dimensional proton nuclear magnetic resonance (2D 1H-NMR) spectroscopy and distance geometry calculations. Unambiguous NMR assignments of protons have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints obtained from the NMR data have been utilized in a distance geometry algorithm to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that, while in water Kassinin prefers to be in an extended chain conformation, in the presence of perdeuterated dodecylphosphocholine (DPC) micelles, a membrane model system, helical conformation is induced in the central core and C-terminal region (K4-M12) of the peptide. N-terminus though less defined also displays some degree of order and a possible turn structure. The conformation adopted by Kassinin in the presence of DPC micelles is consistent with the structural motif typical of neurokinin-1 selective agonists and with that reported for Eledoisin in hydrophobic environment.

Neurons bearing NK(3) tachykinin receptors in the guinea-pig ileum revealed by specific binding of fluorescently labelled agonists.

The localisation of NK(3) tachykinin receptors in guinea-pig ileum was studied using the fluorescently labelled agonists, Cy3. 5-neurokinin A and Cy3.5-kassinin. Binding to nerve cell bodies in the myenteric and submucosal plexuses was visualised using confocal microscopy. Binding to NK(1) receptors was blocked by the NK(1) receptor antagonist, CP-99994. NK(3) receptors, demonstrated by binding in the presence of CP-99994, occurred in 72% of myenteric and 38% of submucosal neurons. Colocalisation with other markers was examined to deduce the classes of neurons with NK(3) receptors. In myenteric ganglia, NK(3) receptors were present on the following: 73% of calbindin-immunoreactive (IR) intrinsic primary afferent neurons, 63% of calretinin-IR excitatory motor neurons and ascending interneurons, 63% of nitric oxide synthase-IR inhibitory motor neurons and descending interneurons, 79% of strongly neuropeptide Y (NPY)-IR secretomotor neurons, 67% of weakly NPY-IR descending interneurons and motor neurons, and 46% of NK(1) receptor-IR neurons. In submucosal ganglia, NK(3) receptors were on 65% of calretinin-IR secretomotor/vasodilator neurons, 81% of NPY-IR cholinergic secretomotor neurons, 2% of vasoactive intestinal peptide-IR non-cholinergic secretomotor neurons and were completely absent from substance P-IR intrinsic primary afferent neurons. The results support physiological studies suggesting that NK(3) receptors mediate tachykinin transmission between myenteric sensory neurons and to interneurons and/or motor neurons in descending inhibitory and ascending excitatory pathways.

Tachykinins and other biologically active peptides from the skin of the Costa Rican phyllomedusid frog Agalychnis callidryas.

Peptides present in a methanol extract prepared from skin of the Costa Rican frog Agalychnis callidryas of the Phyllomedusinae subfamily were studied by sequence analysis and pharmacological tests. Members of five different peptide families-tachykinins, bradykinins, caerulein, opioid peptides and sauvagine-were found. In particular, the extract contained a number of tachykinins with the following sequences: Gly-Pro-Pro-Asp-Pro-Asn-Lys-Phe-Ile-Gly-Leu-Met-NH2, Gly-Pro-Pro-Asp-Pro-Asp-Arg(Lys)-Phe-Tyr-Pro-Gly-Met-NH2, pGlu-Pro-Asp-Pro-Asp-Arg-Phe-Tyr-Pro-Gly-Met-NH2, Gly-Pro-Pro-Asp-Pro-Asn-Lys-Phe-Tyr-Pro-Val-Met. The latter three peptides have the unusual C-terminal sequence Pro-Gly(or Val)-Met-NH2 rather than Gly-Leu-Met-NH2 found in many other members of the tachykinin family. The observed amino acid substitutions may be the reason for the marked decrease in the biological activity observed in all in vitro and in vivo tests, even through the spectrum of tachykinin activities was retained. A kassinin-like peptide, with the sequence Gly-Pro-Pro-Asp-Pro-Asn-Lys-Phe-Ile-Gly-Leu-Met-NH2, was also found in the A. callidryas skin. While kassinin has a much higher affinity for NK-3 than for NK-1 receptors, the opposite is true for this A. callidryas peptide. The extract from A. callidryas skin also contained a new caerulein (pGlu-Asp-Tyr(HSO3)-Lys-Gly-Trp-Met-Asp-Phe-NH2) and a phyllokinin (Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg-Ile-Tyr), as well as the opioid peptides dermorphin and [Hyp6]dermorphin, both previously isolated from different Phyllomedusa species.