Development of Cu(ii)-specific peptide shuttles capable of preventing Cu-amyloid beta toxicity and importing bioavailable Cu into cells

Copper (Cu) in its ionic forms is an essential element for mammals and its homeostasis is tightly controlled. Accordingly, Cu-dyshomeostasis can be lethal as is the case in the well-established genetic Wilson's and Menkes diseases. In Alzheimer's disease (AD), Cu-accumulation occurs in amyloid plaques, where it is bound to the amyloid-beta peptide (A beta). In vitro, Cu-A beta is competent to catalyze the production of reactive oxygen species (ROS) in the presence of ascorbate under aerobic conditions, and hence Cu-A beta is believed to contribute to the oxidative stress in AD. Several molecules that can recover extracellular Cu from A beta and transport it back into cells with beneficial effects in cell culture and transgenic AD models were identified. However, all the Cu-shuttles currently available are not satisfactory due to various potential limitations including ion selectivity and toxicity. Hence, we designed a novel peptide-based Cu shuttle with the following properties: (i) it contains a Cu(ii)-binding motif that is very selective to Cu(ii) over all other essential metal ions; (ii) it is tagged with a fluorophore sensitive to Cu(ii)-binding and release; (iii) it is made of a peptide platform, which is very versatile to add new functions. The work presented here reports on the characterization of AKH-alpha R5W4(NBD), which is able to transport Cu ions selectively into PC12 cells and the imported Cu appeared bioavailable, likely via reductive release induced by glutathione. Moreover, AKH-alpha R5W4(NBD) was able to withdraw Cu from the A beta(1-16) peptide and consequently inhibited the Cu-A beta based reactive oxygen species production and related cell toxicity. Hence, AKH-alpha R5W4(NBD) could be a valuable new tool for Cu-transport into cells and suitable for mechanistic studies in cell culture, with potential applications in restoring Cu-homeostasis in Cu-related diseases such as AD.

Automated summary

Copper homeostasis is crucial for mammal's health and its dysregulation is implicated in diseases like Alzheimer's. In this study, a novel peptide-based copper shuttle is designed, which can selectively transport copper ions into cells and remove copper from amyloid plaques, inhibiting oxidative stress and cell toxicity. This mechanism shows potential applications in treating copper-related diseases.