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

ABSTRACT: 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^IIAβ_4-16, Cu^IIAβ_4-9, and also Cu^IIAβ_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^IIAβ_4-16, Cu^IIAβ_4-9, Cu^IIAβ_12-16, and Cu^IIAβ_1-16 with reduced glutathione (GSH) under aerobic and anaerobic conditions using absorption spectroscopy and mass spectrometry. We found Cu^IIAβ_4-16 and Cu^IIAβ_4-9 to be strongly resistant to reduction and concomitant formation of Cu(I)-GSH complexes, with reaction times ∼10 h, while Cu^IIAβ_12-16 was reduced within minutes and Cu^IIAβ_1-16 within seconds of incubation. Upon GSH exhaustion by molecular oxygen, the Cu^IIAβ 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.

SUBMITTER: Stefaniak E

PROVIDER: S-EPMC7588031 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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

Stefaniak Ewelina E Płonka Dawid D Szczerba Paulina P Wezynfeld Nina E NE Bal Wojciech W

Inorganic chemistry 20200326 7

Aβ4-42 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 CuIIAβ4-16, CuIIAβ4-9, and also CuIIAβ12-16, all with nearly femtomolar affinities at neutra ...[more]

PMID: 32212682

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Project description:Misfolding of the amyloid-β peptide (Aβ) and its subsequent aggregation into toxic oligomers is one of the leading causes of Alzheimer's disease (AD). As a therapeutic approach, cyclic peptides have been modified in many ways and developed as a potential class of amyloid aggregation inhibitors. Head-to-tail cyclic peptides with alternating d, l amino acids inhibit amyloid aggregation significantly. On the other hand, excess deposition of copper, iron, and zinc enhances amyloid aggregation. Dysregulation of these metal ions in the brain triggers aggregation by binding to the Aβ peptide. Therefore, specific metal chelators have been developed for disrupting the Aβ-metal complex, thereby reducing toxicity and restoring metal ion homeostasis. Herein, we report the development of a head-to-tail cyclic peptidomimetic with a copper chelating ligand attached. The designed peptidomimetic inhibits amyloid aggregation significantly in a two-fold molar ratio to the Aβ peptide, as confirmed by the thioflavin T (ThT) fluorescence assay, dynamic light scattering (DLS), transmission electron microscopy (TEM), and Congo-red stained birefringence studies. The chelating ligand attached to the cyclic peptide binds efficiently to Cu2+ but weakly to Zn2+ and Fe2+, thereby exhibiting profound selectivity, probed using UV-visible spectroscopy, thioflavin T (ThT) fluorescence assay, tyrosine (TYR10) fluorescence assay, isothermal titration calorimetry (ITC) and transmission electron microscopy (TEM). The non-toxicity of the designed peptidomimetics and their ability to reduce aggregating Aβ-fragment induced cytotoxicity was confirmed by the MTT assay on the mouse neuronal cell line. Further, the molecular interaction between the peptidomimetics and the Aβ-fragment was confirmed by Förster resonance energy transfer (FRET) studies using fluorescently labeled analogs. Cytotoxicity and cell internalization were also confirmed. A preliminary mechanistic investigation indicates that the peptidomimetic works by a synergistic effect of conformational restriction and metal sequestration. Such peptidomimetics can shed light on the mechanism of aggregation and a novel therapeutic approach.

Dataset Information

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

Publications

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

Similar Datasets

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