The tachykinin peptide neurokinin B binds copper(I) and silver(I) and undergoes quasi-reversible electrochemistry: Towards a new function for the peptide in the brain

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Highlights

Abstract

The tachykinin neuropeptide family, which includes substance P and neurokinin B, is involved in a wide array of biological functions. Among these is the ability to protect against the neurotoxic processes in Alzheimer’s Disease, but the mechanisms driving neuroprotection remain unclear. Dysregulation of metal ions, particularly copper, iron and zinc is a common feature of Alzheimer’s Disease, and other amyloidogenic disorders. Copper is known to be released from neurons and recent work has shown that some tachykinins can bind Cu(II) ions, and that neurokinin B can inhibit copper uptake into astrocytes. We have now examined whether neurokinin B is capable of binding Cu(I), which is predicted to be available in the synapse. Using a combination of spectroscopic techniques including cyclic voltammetry and magnetic resonance we show that neurokinin B can bind Cu(I) either directly from added CuCl or by reduction of Cu(II)-bound neurokinin B. The results showed that the Cu(I) binding site differs greatly to that of Cu(II) and involves thioether coordination via Met2 and Met10 and an imidazole nitrogen ligand from His3. The Cu(I) coordination is also different to the site adopted by Ag(I). During changes in oxidation state, copper remains bound to neurokinin B despite large changes to the inner coordination sphere. We predict that neurokinin B may be involved in synaptic copper homeostasis.

Introduction

Neurokinin B (NKB, sequence DMHDFFVGLM-NH2), along with neurokinin A (NKA) and substance P (SP), are the most recognized members of the endogenous mammalian tachykinin (TK) family of neuropeptides. TKs have various roles throughout the central and peripheral nervous system and act as neurotransmitters, neuromodulators and neuroprotectants (Pieri et al., 2005, Raffa, 1998, Stacey et al., 2002). These neuropeptides share a conserved C-terminal sequence (Phe-Xxx-Gly-Leu-Met-NH2 where Xxx = Phe, Val, Tyr or Ile), which is essential for receptor binding. SP, NKA and NKB exhibit preferential binding to neurokinin-1 (NK1), neurokinin-2 (NK2) and neurokinin-3 (NK3) G protein-coupled receptors, respectively (Khawaja and Rogers, 1996, Pennefather et al., 2004).

The neuroprotective capabilities of TKs have been highlighted by their possible role in protecting against Alzheimer’s Disease (AD). Intriguingly, both SP and NKB can partially reverse the neurotoxic actions of the amyloid β (Aβ1-40) peptide on hippocampal neurons (Yankner et al., 1990), yet it is not understood how this occurs. Recent studies suggest that copper may form the link between AD and some tachykinins, but this relationship is complex and possibly multifactorial. Studies have shown that NKB, NKA and extended forms of NKA, namely neuropeptide γ and neuropeptide K, are able to bind Cu(II) (Blaszak et al., 2013, Kowalik-Jankowska et al., 2010, Pietruszka et al., 2011, Russino et al., 2013). This feature may be one of the mechanisms affording protection against AD because the peptides could compete with neurotoxic Aβ peptides for copper which may in turn limit the copper-dependent aggregation of the Aβ peptides. Copper is released from neurons into the extracellular space where it may interact with Aβ peptides and promote misfolding and aggregation (Hopt et al., 2003, Schlief et al., 2005, Schlief and Gitlin, 2006). AD plaques contain high copper concentrations (>400 μM) while the surrounding neuronal cells are depleted of copper (Donnelly et al., 2007, Hung et al., 2010) suggesting that plaque formation contributes to metal dysregulation.

TKs may also be intimately involved with local oxidative stress, which is important because the generation of reactive oxygen species (ROS) is a key feature of AD (Ansari and Scheff, 2010, Kozlowski et al., 2009). This may occur by direct effects of NKs on neurons. In a neuroprotective capacity, recent studies have shown that NKB can increase the activity of superoxide dismutase (SOD; an antioxidant), which is normally down regulated in AD (Maier and Chan, 2002). However, it may also involve TK binding of copper. In AD and some other neurodegenerative disorders, ROS may be generated as a result of the dysregulation of copper (Duce and Bush, 2010, Kozlowski et al., 2009, Minicozzi et al., 2008, White et al., 2006). NKA forms pH dependent monomeric and dimeric four- or five-coordinate complexes with Cu(II) (Kowalik-Jankowska et al., 2010). In this case, rather than being neuroprotective the study showed that metal-catalyzed oxidation of NKA residues bound to Cu(II), via reaction with hydrogen peroxide (H2O2), not only destroyed the peptide’s primary and secondary structure but could potentially contribute to ROS generation.

Cu(II) binding to NKB has been shown to result in the formation of an unusual binary complex, [CuII(NKB)2], with a coordination sphere predicted to consist of the N-terminal amine and the His3 imidazole nitrogen from each monomer (Russino et al., 2013). Despite forming the duplex, NKB still retained functionality in terms of Ca2+ activation in astrocytes (Russino et al., 2013). Furthermore, NKB was able to restrict the uptake of copper by astrocytes in vitro and this inhibited the ability of ‘free’ copper to dysregulate intracellular calcium levels (Russino et al., 2013). Given that NKB can bind copper and restrict copper uptake we predict that it is possible, that under normal conditions, NKB has a role in synaptic copper homeostasis. A complexity associated with this concept is the oxidation state of copper itself and how this might affect binding and the functionality of NKs. Although under most conditions extracellular copper is considered to exist in the oxidized Cu(II) state, conditions in a synapse are likely to be controlled and quite different to the systemic extracellular spaces. For instance, the synapse is high in the biological reductants ascorbate and glutathione (Rebec et al., 2005, Rice, 2000, Rice and Russo-Menna, 1998), and is often relatively hypoxic compared to normal conditions (Erecinska and Silver, 2001). Changes in the synaptic redox state are known to affect the function of several receptors that contain extracellular cysteine ligands (Sullivan et al., 1994). It is therefore highly likely that within neuronal synapses reduced copper (Cu(I)) is present. Support for this concept comes from the loading of copper into vesicles for release into the synapse by hippocampal neurons. These vesicles are loaded by the Menkes ATPase, ATP7a, which only shuttles Cu(I), and it is presumably Cu(I) that is released into the synapse (Schlief and Gitlin, 2006). The interaction of Cu(I) with synaptic components has often been overlooked, yet proteins such as the prion protein and the Alzheimer’s-related amyloid precursor protein have been shown to bind Cu(I) (Badrick and Jones, 2009, Shearer and Soh, 2007). The ability of TKs to bind Cu(I) has not yet been investigated. Although NKB has been shown to bind Cu(II), it is highly probable that NKB is also able to bind Cu(I) as it contains Met and His amino acids that are suitable for Cu(I) coordination. An ability to bind copper as Cu(I) as well as Cu(II) would support the potential role for NKB in synaptic copper homeostasis. If such complexation were to occur, any potential redox cycling may contribute to the production of ROS through Fenton reactions which have been implicated in neurodegeneration. In this work we explore the electrochemistry of copper-bound NKB, and elucidate details of the coordination of Cu(I) and Ag(I).

Section snippets

Materials and methods

NKB (Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2) was purchased from Auspep PL (Melbourne, Australia) and used without further purification. The concentration of NKB was calculated by taking into account the supplier’s estimated H2O content (25%) and confirmed using the extinction coefficient of the two phenylalanine residues. Deionised water (18 MΩ) was used for all solution preparations. Cu(I) experiments required that all solutions involved (acetonitrile (ACN) and sodium phosphate buffer (NaPi

Copper-bound NKB undergoes quasi-reversible redox cycling

Previous work has established that NKB can bind Cu(II) as a [CuII(NKB)2] complex. Given that the peptide contains a His and two Met residues within a short sequence of only ten amino acids, we hypothesized that the peptide would also be able to bind Cu(I). Cu(I) is a d10 metal ion and prefers softer ligands such as sulfur over harder nitrogen and oxygen Cu(II) ligands. Recent work has shown that thioether coordination is able to stabilize Cu(I) at higher potentials than the reducing

Discussion

The important role of Cu in general biology has been appreciated for many years, yet the metal’s function in the brain, and particularly once it is released by neurons, is not clear. Most research in this area has focused on possible pathological roles for extracellular copper, such as in Alzheimer’s and Parkinson’s diseases and in prion disorders. The fact that copper is released from synaptic vesicles (Barnea et al., 1989, Dodani et al., 2011, Hartter and Barnea, 1988, Schlief et al., 2005)

Conclusion

Although TKs have many roles in the CNS from activation of the immune system to regulators of pain transmission to key molecules in reproductive function, our results suggest that NKB also has a role in synaptic Cu homeostasis where neuronal control of peptide and Cu release can fine tune where and how the metal ion is used. Intriguingly, this may be a role for not only NKB, but perhaps also for other tachykinins, particularly NKA which is expressed in the brain and is known to bind copper (

Acknowledgements

UWS is thanked for financial support.

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