Copper Transporters? Glutathione Reactivity of Products of Cu-Aß Digestion by Neprilysin.

ABSTRACT

Aß4-42 is the major subspecies of Aß 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 CuIIAß4-16, CuIIAß4-9, and also CuIIAß12-16, 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 CuIIAß4-16, CuIIAß4-9, CuIIAß12-16, and CuIIAß1-16 with reduced glutathione (GSH) under aerobic and anaerobic conditions using absorption spectroscopy and mass spectrometry. We found CuIIAß4-16 and CuIIAß4-9 to be strongly resistant to reduction and concomitant formation of Cu(I)-GSH complexes, with reaction times ∼10 h, while CuIIAß12-16 was reduced within minutes and CuIIAß1-16 within seconds of incubation. Upon GSH exhaustion by molecular oxygen, the CuIIAß complexes were reformed with no concomitant oxidative damage to peptides. These finding reinforce the concept of Aß4-x peptides as physiological trafficking partners of brain copper.