Investigating the role of phenylalanine residues for amyloid formation of the neuropeptide neurokinin B.

Neurokinin B (NKB) is a tachykinin peptide that has diverse roles in biology, including in human reproductive development. Cellular processing of this peptide is thought to involve formation of a dense core vesicle during transit through the regulated secretory pathway. The ability of NKB to rapidly form an amyloid can contribute to formation of the secretory granule but features that support amyloid formation of NKB are not well understood. NKB contains a diphenylalanine sequence well recognised as an important motif for self-assembly of other peptides including amyloid ß. Using mutations of the diphenylalanine motif we show that this motif in NKB is necessary for amyloid formation, and it is the unique combination of aromaticity and hydrophobicity of phenylalanine that is crucial for aggregation. Using disulfide cross-linking we propose that phenylalanine at sequence position 6 is important for stabilising inter-sheet interactions in the NKB amyloid fibril. Although having a highly conserved sequence, the NKB peptide from zebrafish only contains a single phenylalanine and does not fibrillise as extensively as mammalian NKB. Analysis of self-assembly of NKB-like peptides from different species may help in elucidating their biological roles. Taken together, this work shows that mammalian NKB has evolved, within only 10 residues, a sequence optimised for rapid self-assembly, whilst also containing residues for metal-binding, receptor binding and receptor discrimination.

Spectral Phasor Analysis of Nile Red Identifies Membrane Microenvironment Changes in the Presence of Amyloid Peptides.

The interaction of protein and peptide amyloid oligomers with membranes is thought to be one of the mechanisms contributing to cellular toxicity. However, techniques to study these interactions in the complex membrane environment of live cells are lacking. Spectral phasor analysis is a recently developed biophysical technique that can enable visualisation and analysis of membrane-associated fluorescent dyes. When the spectral profile of these dyes changes as a result of changes to the membrane microenvironment, spectral phasor analysis can localise those changes to discrete membrane regions. In this study, we investigated whether spectral phasor analysis could detect changes in the membrane microenvironment of live cells in the presence of fibrillar aggregates of the disease-related Aß42 peptide or the functional amyloid neurokinin B. Our results show that the fibrils cause distinct changes to the microenvironment of nile red associated with both the plasma and the nuclear membrane. We attribute these shifts in nile red spectral properties to changes in membrane fluidity. Results from this work suggest that cells have mechanisms to avoid or control membrane interactions arising from functional amyloids which have implications for how these peptides are stored in dense core vesicles. Furthermore, the work highlights the utility of spectral phasor analysis to monitor microenvironment changes to fluorescent probes in live cells.

Essential Role of Histidine for Rapid Copper(II)-Mediated Disassembly of Neurokinin B Amyloid.

Neurokinin B is a tachykinin peptide involved in a diverse range of neuronal functions. It rapidly forms an amyloid, which is considered physiologically important for efficient packing into dense core secretory vesicles within hypothalamic neurons. Disassembly of the amyloid is thought to require the presence of copper ions, which interact with histidine at the third position in the peptide sequence. However, it is unclear how the histidine is involved in the amyloid structure and why copper coordination can trigger disassembly. In this work, we demonstrate that histidine contributes to the amyloid structure via π-stacking interactions with nearby phenylalanine residues. The ability of neurokinin B to form an amyloid is dependent on any aromatic residue at the third position in the sequence; however, only the presence of histidine leads to both amyloid formation and rapid copper-induced disassembly.

Copper ions trigger disassembly of neurokinin B functional amyloid and inhibit de novo assembly.

The formation of amyloid is considered an intrinsic ability of most polypeptides. It is a structure adopted by many neuropeptides and neurohormones during the formation of dense core vesicles in secretory cells, yet the mechanisms mediating assembly and disassembly of these amyloids remain unclear. Neurokinin B is a neuropeptide thought to form an amyloid in secretory cells. It is known to coordinate copper, but the physiological significance of metal binding is not known. In this work we explored the amyloid formation of neurokinin B and the impact that metals had on the aggregation behaviour. We show that the production of neurokinin B amyloid is dependent on the phosphate concentration, the pH and the presence of a histidine at position 3 in the primary sequence. Copper(II) and nickel(II) coordination to the peptide, which requires the histidine imidazole group, completely inhibits amyloid formation, whereas zinc(II) slows, but does not inhibit fibrillogenesis. Furthermore, we show that copper(II) can rapidly disassemble preformed neurokinin B amyloid. This work identifies a role for copper in neurokinin B structure and reveals a mechanism for amyloid assembly and disassembly dependent on metal coordination.

A gonadotropin-releasing hormone type neuropeptide with a high affinity binding site for copper(ii) and nickel(ii).

In vertebrates gonadotropin-releasing hormone I (GnRH-I) is a key regulator of reproductive development and function. The receptor-binding activity of human GnRH-I can be modified by the presence of divalent copper. Thus, copper binding to N-terminal amino acids in GnRH-I induces structural changes that influence receptor interactions and downstream intracellular signalling cascades. It is not known if copper-binding is restricted to human GnRH-I or if it is also a feature of GnRH-type peptides that have been identified in other taxa. To investigate this, we have characterised copper binding to a recently discovered GnRH-type peptide from the starfish Asterias rubens (ArGnRH). Using a range of spectroscopic and biophysical techniques we show that this peptide can bind copper(ii) and nickel(ii). Copper(ii) is bound in a square-planar, high-affinity (Kd ∼ 10-12 M) site incorporating four nitrogen donor atoms from a histidine imidazole group, two amides and the N-terminal amine group. The ArGnRH copper affinity and geometry are quite different to GnRH-I suggesting the copper sites have evolved to suit the environment the peptides are exposed to. By comparing the copper binding sites in ArGnRH and human GnRH-I and conducting a phylogenetic analysis of GnRH-type peptide sequences from a range of species, we predict that copper-binding is an evolutionarily ancient feature of GnRH-type peptides that has been retained, modified or lost in different lineages.