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 neuro-peptide 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

[In Press] 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 A beta(42) 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