Copper Transporters? Glutathione Reactivity of Products of Cu–Aβ Digestion by Neprilysin

[Image: see text] Aβ(4–42) is the major subspecies of Aβ peptides characterized by avid Cu(II) binding via the ATCUN/NTS motif. It is thought to be produced in vivo proteolytically by neprilysin, but in vitro experiments in the presence of Cu(II) ions indicated preferable formation of C-terminally truncated ATCUN/NTS species including Cu(II)Aβ(4–16), Cu(II)Aβ(4–9), and also Cu(II)Aβ(12–16), all with nearly femtomolar affinities at neutral pH. Such small complexes may serve as shuttles for copper clearance from extracellular brain spaces, on condition they could survive intracellular conditions upon crossing biological barriers. In order to ascertain such possibility, we studied the reactions of Cu(II)Aβ(4–16), Cu(II)Aβ(4–9), Cu(II)Aβ(12–16), and Cu(II)Aβ(1–16) with reduced glutathione (GSH) under aerobic and anaerobic conditions using absorption spectroscopy and mass spectrometry. We found Cu(II)Aβ(4–16) and Cu(II)Aβ(4–9) to be strongly resistant to reduction and concomitant formation of Cu(I)–GSH complexes, with reaction times ∼10 h, while Cu(II)Aβ(12–16) was reduced within minutes and Cu(II)Aβ(1–16) within seconds of incubation. Upon GSH exhaustion by molecular oxygen, the Cu(II)Aβ complexes were reformed with no concomitant oxidative damage to peptides. These finding reinforce the concept of Aβ(4–x) peptides as physiological trafficking partners of brain copper.