The Tachykinin Peptide Neurokinin B Binds Copper Forming an Unusual [Cu^II(NKB)₂] Complex and Inhibits Copper Uptake into 1321N1 Astrocytoma Cells

Abstract

Neurokinin B (NKB) is a member of the tachykinin family of neuropeptides that have neuroinflammatory, neuroimmunological, and neuroprotective functions. In a neuroprotective role, tachykinins can help protect cells against the neurotoxic processes observed in Alzheimer's disease. A change in copper homeostasis is a clear feature of Alzheimer's disease, and the dysregulation may be a contributory factor in toxicity. Copper has recently been shown to interact with neurokinin A and neuropeptide γ and can lead to generation of reactive oxygen species and peptide degradation, which suggests that copper may have a place in tachykinin function and potentially misfunction. To explore this, we have utilized a range of spectroscopic techniques to show that NKB, but not substance P, can bind Cu II in an unusual [Cu II (NKB) 2 ] neutral complex that utilizes two N-terminal amine and two imidazole nitrogen ligands (from each molecule of NKB) and the binding substantially alters the structure of the peptide. Using 1321N1 astrocytoma cells, we show that copper can enter the cells and subsequently open plasma membrane calcium channels but when bound to neurokinin B copper ion uptake is inhibited. This data suggests a novel role for neurokinin B in protecting cells against copper-induced calcium changes and implicates the peptide in synaptic copper homeostasis. KEYWORDS: Neurokinin B, tachykinin, copper, calcium, neurodegeneration, substance P, Fura2, EPR N eurokinin B (NKB, neuromedin K) is a 10 residue peptide that belongs to the tachykinin family of neuropeptides. Other members include the mammalian peptides substance P (SP), neurokinin A (NKA), the Nterminally extended forms of NKA neuropeptide K and neuropeptide γ, and hemokinin.1 The family is characterized by the presence of a common C-terminal sequence FxGLM-NH 2 , which is thought to be important for receptor binding and activation.2 The biological activity of the tachykinin peptides lies with their ability to bind to G protein-coupled receptors and to mediate intracellular calcium release via several signaling pathways, including a G q/11 -coupled pathway, which involves the downstream generation of the second messengers diacylglycerol and IP 3 .2,3 SP, NKA, and NKB bind to neurokinin receptors NK1-R, NK2-R, and NK3-R, and although each peptide can bind to all the receptors, each has a preference for a particular receptor. Thus SP has a preference for NK1-R, NKA for NK2-R, and NKB for NK3-R. The structure of the peptides bound to their receptors is unclear; however, a recent modeling study looking at the interaction of NKB with NK3-R has highlighted not only how the C-terminal conserved domain directs receptor binding but also how the Nterminal residues may be involved in mediating receptor preference. 4 Functionally, the tachykinins are involved in a diverse range of cellular signaling processes including roles in neuroprotection, neuroinflammation, and neurotrophic pathways.

N‐Terminal Cu‐Binding Motifs (Xxx‐Zzz‐His, Xxx‐His) and Their Derivatives: Chemistry, Biology and Medicinal Applications

“…A multitude of proteins and peptides have the N‐terminal motif XZH. This includes serum albumins of humans and most mammals, salivary histatins, Ctr1, neurokinin B, hepcidin, sperm protamine 2 of humans and several other mammals, and others. However, in humans only serum albumin (HSA) clearly binds Cu II under physiological conditions.…”

Neuropeptides Exert Neuroprotective Effects in Alzheimer's Disease

“…It is known that the dysfunction of metal ions, such as copper is a feature of Alzheimer's disease. Neurokinin B protects against copper-induced calcium channel opening (Russino et al, 2013), as well as the synaptic homeostasis (Grosas et al, 2014).…”

Tachykinin: recent developments and novel roles in health and disease

“…The inhibition of copper uptake appears to protect against copper-induced intracellular calcium fluctuations. Although SP does not exert any copper-binding activity, a novel protective role of NKB in AD was recently demonstrated (157); that is, two NKB molecules can bind a copper ion, preventing copper from entering astrocytes. In the cerebrospinal fluid of HD, free copper ion concentration was shown to increase, while copper/superoxide dismutase (SOD) activity decreased according to the stage of the disease (158).…”