Studies of Copper - ATPases

Copper is an integral cofactor for numerous enzymes in various cell processes, including oxidative metabolism, neurotransmitter synthesis, free radical detoxification, and iron uptake. Among the proteins known to be important in the regulation of copper levels in epithelial cells are two Cu-transporting membrane ATPases, ATP7A (Menkes protein) and ATP7B (Wilson protein). They share ~57% amino acid identity. Menkes protein is widely distributed (eg, intestine, connective tissue, brain), whereas Wilson is expressed predominantly in the liver, but also kidney and some parts of the brain. These ATPases transport Cu across a membrane from the cytoplasm to an “outside” space and they sense Cu levels in the cytoplasm. ATP7A and ATP7B are both present predominantly in the Golgi region when intracellular copper levels are low (Figure 1), but ATP7A redistributes to the PM at high copper levels, while ATP7B moves to vesicles at the periphery under the same conditions (Figure 2).

Figure 1. At low to normal Cu levels, ATP7B resides in the Golgi region in transfected CHO cells. Left panel, ATP7B;

middle panel, gamma-adaptin; right panel, merged images.

 

Figure 2.  Addition of copper to the growth medium causes a shift in ATP7B localization from the Golgi network to cytoplasmic vesicles.

Left panel, ATP7B; middle, gamma-adaptin; right, merged images.   

Hepatic copper homeostasis, mediated by ATP7B, is dependent on both copper transport activity of the ATPase and its correct intracellular localization. This means that at different copper levels, ATP7B plays two  different roles. One role is biosynthetic, delivering copper to apo-ceruloplasmin within the Golgi network. The other role is to transport excess copper out of the cell and into the bile canaliculus for subsequent  excretion from the body via the digestive tract.

 

Wilson disease is an inherited disease of copper excess that is caused by mutations in ATP7B. Wilson disease can present in childhood with hepatotoxicity due to copper overload. Despite zinc or copper chelation therapy, the prognosis for young people is liver transplantation. Therefore, learning more about the molecular mechanisms by which cytoplasmic copper regulates the intracellular dynamics of normal Wilson protein would be a step toward developing new molecular therapies that might correct and/or reverse the course of this devastating disease.

To date, most studies of ATP7A and ATP7B have been carried out in non-physiological cell systems (eg, the unpolarized CHO cells). We are focusing on ATP7B using a polarized hepatic cell line developed and characterized in our laboratory.  I will study the copper-dependent dynamics of the protein, to understand the molecular mechanisms by which cytoplasmic Cu levels regulate the intracellular dynamics of the Cu-ATPase.