Acting on protein unfolding could be a promising alternative in developing new therapeutic strategies for patients with Wilson disease, according to a study published in Cells.

ATP7B is an essential membrane protein responsible for transporting copper across both sides of the lipid bilayer. It transports proteins between the Golgi apparatus, to deliver copper for the maturation of proteins like ceruloplasmin, and the bile canaliculi, to carry out the excretion process into the bile. 

Wilson disease is an autosomal-recessive genetic disorder resulting from mutations in the ATP7B gene. Generally, the secretion of copper into the bile canaliculi is impaired, leading to the accumulation of copper in the liver and brain. Bis-choline-tetrathiomolybdate, another copper chelator possessing a high affinity for copper(I), has been manufactured and successfully tested in phase 2 clinical trials.

Recent studies have shown that HepG2/C3, a hepatocarcinoma cell line, can be developed three-dimensionally to automatically form bile canaliculi structures functional for the excretion of organic molecules, silver, and copper. Thus, the abovementioned cellular system could be relevant to Wilson disease research.

Read more about Wilson disease clinical trials

The research team in the present study applied the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) strategy to design a HepG2/C3a cell line by impeding the ATP7B coding region. Response to copper in Wilson disease hepatocytes was then compared with that in the parental cell line. 

The study revealed that the sensitivity to severe copper stress was reduced by around 100 µM, corresponding to 20% of the lethal dose for 50% of the cells. Moreover, the cellular responses occurring under moderate copper stress revealed the vital role of ATP7B at a few micromolar copper concentrations.

Notably, metallothionein (Met) expression and glutathione (GSH) production started at a lower copper concentration but reached the same magnitude as that in HepG2/C3a cells. In contrast, heat shock protein family A member 6 (HSPA6) expression was highly intensified in the ATP7B‑deficient cells. The above results highlighted the importance of copper‑induced protein misfolding in the Wilson disease-like hepatocytes. At the same time, other stresses were not altered to that extent. 

“In terms of the stress response, our data showed that protein misfolding is the main stress induced by [copper], drastically exacerbated in ATP7B‑defcient cells,” the authors wrote. “Indeed, our data showed a huge overexpression of HSPA6 that is known to be expressed under severe stress conditions. Therefore, acting on protein unfolding represents a promising alternative to developing new therapeutic approaches for Wilson patients.”

The authors concluded that ATP7B‑deficient cell lines could serve as valuable tools to better understand copper homeostasis.


Charbonnier P, Chovelon B, Ravelet C, Ngo TD, Chevallet M, Deniaud A. ATP7B-deficient hepatocytes reveal the importance of protein misfolding induced at low copper concentration. Cells. 2022;11(21):3400. doi:10.3390/cells11213400